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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
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Email: schalk@unf.edu
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Biochemistry

  • Publisher: American Chemical Society
  • FAD Code: BCHM
  • CODEN: BICHAW
  • ISSN: 0006-2960
  • Abbreviation: Biochemistry
  • DOI Prefix: 10.1021/bi
  • Language: English
  • Comments: Fulltext from 1962 V1

Citations 29

"Reaction-induced Infrared Difference Spectroscopy For The Study Of Protein Reaction Mechanisms"
Biochemistry 2001 Volume 40, Issue 7 Pages 1875-1883
Christian Zscherp and Andreas Barth

Abstract: This paper reviews state-of-the-art reaction-induced infrared difference spectroscopy of proteins. This technique enables detailed characterization of enzyme function on the level of single bonds of proteins, cofactors, or substrates. The following methods to initiate a reaction in the infrared sample are discussed: (i) light-induced difference spectroscopy, (ii) attenuated total reflection with buffer exchange, (iii) the infrared variant of stopped and continuous flow, (iv) temperature and pressure jump, (v) photolytical release of effector substances from caged compounds, (vi) equilibrium electrochemistry, and (vii) photoreduction. Illustrating applications are given including hot topics from the fields of bioenergetics, protein folding, and molecule-protein interaction.

"Transient-state Kinetic Analysis Of Saccharomyces Cerevisiae MyristoylCoA : Protein N-myristoyltransferase Reveals That A Step After Chemical Transformation Is Rate Limiting"
Biochemistry 2000 Volume 39, Issue 51 Pages 15807-15816
Thalia A. Farazi, Jill K. Manchester, and Jeffrey I. Gordon

Abstract: MyristoylCoA:protein N-myristoyltransferase is a member of the superfamily of GCN5-related N-acetyltransferases and catalyzes the covalent attachment of myristate to the N-terminal Gly residue of proteins with diverse functions. Saccharomyces cerevisiae Nmt1p is a monomeric protein with an ordered bi-bi reaction mechanism: myristoylCoA is bound prior to peptide substrate; after catalysis, CoA is released followed by myristoylpeptide. Analysis of the X-ray structure of Nmt1p with bound substrate analogues indicates that the active site contains an oxyanion hole and a catalytic base and that catalysis proceeds through the nucleophilic addition-elimination mechanism. To determine the rate-limiting step in the enzyme reaction, pre-steady-state kinetic analyzes were performed using a new, sensitive nonradioactive assay that detects CoA. Multiple turnover quenched flow studies disclosed that a step after the chemical transformation limits the overall rate of the reaction. Multiple and single turnover analyzes revealed that the rate for the chemical transformation step is 13.8±0.6 s-1 while the slower steady-state phase is 0.10±0.01 s-1. Stopped flow kinetic studies of substrate acquisition indicated that binding of myristoylCoA to the ape-enzyme occurs through at least a two-step process, with a fast phase rate of 3.2 x 10^8 M-1 s-1 and a slow phase rate of 23±2 s-1 (defined at 5°C). Binding of an octapeptide substrate, representing the N-terminal sequence of a known yeast N-myristoylprotein (Cnb1p), to a binary complex composed of Nmt1p and a nonhydrolyzable myristoylCoA analogue (S-(2-oxo)pentadecylCoA) has a second-order rate constant of 2.1±0.3 x 10^6 M-1 s-1 and a dissociation rate of 26±15 s-1 (defined at 10°C). These results are interpreted in light of the X-ray structures of this enzyme.

"Reactivity Of Horseradish Peroxidase Compound II Toward Substrates: Kinetic Evidence For A Two-step Mechanism"
Biochemistry 2000 Volume 39, Issue 43 Pages 13201-13209
José Neptuno Rodríguez-López, María Angeles Gilabert, José Tudela, Roger N. F. Thorneley, and Francisco García-Cánovas

Abstract: Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2-azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M-1 s-1) prior to rate-limiting electron transfer (k(+6) = 42.1 s-1). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s-1) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.
Rate constants

"Mutational Analysis Of Metallo-β-lactamase CcrA From Bacteroides Fragilis"
Biochemistry 2000 Volume 39, Issue 37 Pages 11330-11339
Margaret P. Yanchak, Rebecca A. Taylor, and Michael W. Crowder

Abstract: In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-β-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyzes, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat) suggesting that Asn193 does not play a large role in catalysis. However, stopped flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.

"Changes In The Apomyoglobin Folding Pathway Caused By Mutation Of The Distal Histidine Residue"
Biochemistry 2000 Volume 39, Issue 37 Pages 11227-11237
Carlos Garcia, Chiaki Nishimura, Silvia Cavagnero, H. Jane Dyson, and Peter E. Wright

Abstract: Factors governing the folding pathways and the stability of apomyoglobin have been examined by replacing the distal histidine at position 64 with phenylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significantly more stable than wild-type apoMb. Kinetic refolding studies of this variant also show a significant difference from wild-type apoMb. The amplitude of the burst phase ellipticity in stopped-flow CD measurements is increased over that of wild-type, an indication that the secondary structure content of the earliest kinetic intermediate is greater in the mutant than in the wild-type protein. In addition, the overall rate of folding is markedly increased. Hydrogen exchange pulse labeling was used to establish the structure of the initial intermediate formed during the burst phase of the H64F mutant. NMR analysis of the samples obtained at different refolding times indicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic intermediate. Replacement of the polar distal histidine residue with a nonpolar residue of similar size and shape appears to stabilize the E helix in the early stages of folding due to improved hydrophobic packing. The presence of a hydrophilic histidine at position 64 thus exacts a price in the stability and folding efficiency of the apoprotein, but this residue is nevertheless highly conserved among myoglobins due to its importance in function.

"Stopped-flow And Steady-state Study Of The Diphenolase Activity Of Mushroom Tyrosinase"
Biochemistry 2000 Volume 39, Issue 34 Pages 10497-10506
Jos&eacute; Neptuno Rodr&iacute;guez-L&oacute;pez, Lorena G. Fenoll, Pedro Antonio Garc&iacute;a-Ruiz, Ram&oacute;n Var&oacute;n, Jos&eacute; Tudela, Roger N. F. Thorneley, and Francisco Garc&iacute;a-C&aacute;novas

Abstract: The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10^7 M-1 s-1, which is similar to that reported for hemocyanins [(1.3 x 10^6) - (5.7 x 10^7) M-1 s-1]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K-D(O2) = 46.6 µM, which is similar to the value (K-D(O2) = 90 µM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M-1 s-1) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M-1 s-1). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.

"Insights Into The HER-2 Receptor Tyrosine Kinase Mechanism And Substrate Specificity Using A Transient Kinetic Analysis"
Biochemistry 2000 Volume 39, Issue 32 Pages 9786-9803
Amy Y. Jan, Eric F. Johnson, A. John Diamonti, Kermit L. Carraway III, and Karen S. Anderson

Abstract: The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide k(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pn-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.

"The Role Of Glutamic Acid-69 In The Activation Of Citrobacter Freundii Tyrosine Phenol-lyase By Monovalent Cations"
Biochemistry 2000 Volume 39, Issue 29 Pages 8546-8555
Bakthavatsalam Sundararaju, Haoyuan Chen, Steven Shilcutt, and Robert S. Phillips

Abstract: Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K+, NH4+, and Rb+. Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with Ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate Ligation by one of the εoxygens of Glu69 from another chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K+, NH4+, and Rb+, with K-D values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation. Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination. Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K+ Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process. In agreement with this conclusion, a slow increase (k < 0.5 s-1) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K+, Rb+, and NH4+ but not Li+ or Na+. E69D TPL shows no change in fluorescence under these conditions. High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL. This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.

"Effect Of An Alternative Disulfide Bond On The Structure, Stability, And Folding Of Human Lysozyme"
Biochemistry 2000 Volume 39, Issue 12 Pages 3472-3479
Munehito Arai, Patrice Hamel, Eiko Kanaya, Koji Inaka, Kunio Miki, Masakazu Kikuchi, and Kunihiro Kuwajima

Abstract: Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the β-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys63-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W63CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the β-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.

"Reversible And Irreversible Steps In Assembly And Disassembly Of Vesicular Stomatitis Virus: Equilibria And Kinetics Of Dissociation Of Nucleocapsid-M Protein Complexes Assembled In Vivo"
Biochemistry 1998 Volume 37, Issue 2 Pages 439-450
Douglas S. Lyles and Margie O. McKenzie

Abstract: The matrix (M) protein of vesicular stomatitis virus (VSV) condenses the viral nucleoprotein core (nucleocapsid) into a tightly coiled, helical nucleocapsid-M protein (NCM) complex. Using NCM complexes assembled in vivo, the dissociation of M protein was examined by measuring the apparent affinity constants and kinetic constants for M protein binding to NCM complexes immediately after detergent solubilization of the virion envelope. Wild-type VSV strains and viruses with mutations in their M proteins were analyzed using sedimentation and light-scattering assays. At physiological ionic strength, the binding reaction had the characteristics of a dynamic reversible equilibrium. A temperature-sensitive M protein mutant lost the ability of M protein to reversibly dissociate from the nucleocapsid, while a temperature-stable revertant regained the ability to undergo reversible dissociation. In contrast to the results obtained at physiological ionic strength, nucleocapsids stripped of M protein by incubation at high ionic strength (250 mM NaCl) were not able to bind M protein at low ionic strength with the same high affinity seen in NCM complexes assembled in vivo. The effect of incubation at 250 mM NaCl was shown to be due to a change in nucleocapsids rather than a change in soluble M protein. This result supports the idea that nucleocapsids devoid of M protein must undergo a separate step that initiates high-affinity binding of M protein in vivo.
Protein, folding Protein, binding Kinetic

"Kinetics Of Cytochrome C Folding Examined By Hydrogen Exchange And Mass Spectrometry"
Biochemistry 1997 Volume 36, Issue 48 Pages 14992-14999
Houjun Yang and David L. Smith

Abstract: Pulsed hydrogen exchange/mass spectrometry, a new method for studying protein folding, has been used to investigate folding of cytochrome c on the 5 ms to 15 s time scale. Cytochrome c, unfolded in guanidine hydrochloride/D2O, was allowed to refold in a high-speed quenched-flow apparatus and pulse-labeled with protium to identify unfolded regions. Intact, labeled cytochrome c was digested into fragments which were analyzed by HPLC electrospray ionization mass spectrometry to determine the level of deuterium in each fragment. Bimodal distributions of deuterium were found for most segments, indicating that regions represented by these segments were either unfolded or completely folded in the intact polypeptide prior to labeling. This behavior is consistent with cooperative, localized folding which occurs in less than 10 ms in individual molecules. Deuterium levels found in the fragments were normalized to levels found in the same fragments derived from folded cytochrome c, pulse-labeled in the same manner, to indicate the percentage of cytochrome c that was folded. These results show that the N/C-terminal regions fold cooperatively on a time scale extending from less than the mixing time of the apparatus (5 ms) to as long as 15 s, and that the other regions also fold cooperatively. However, these regions do not begin to fold until 30 ms after mixing. In addition to providing new information on cytochrome c folding, these results demonstrate that pulse-hydrogen exchange/mass spectrometry is complementary to NMR in some respects and advantageous in others. Results of this study form the foundation required to extend the pulsed hydrogen exchange approach to folding studies of proteins too large to be analyzed by NMR.
Cytochrome C Mass spectrometry HPLC Kinetic

"Minimizing Nonproductive Substrate Binding: A New Look At Glucoamylase Subsite Affinities"
Biochemistry 1997 Volume 36, Issue 48 Pages 14946-14955
Sateesh K. Natarajan and and Michael R. Sierks

Abstract: A subsite model as proposed by Hiromi [Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1-6] has been applied to various hydrolases including glucoamylase (GA). The model assumes a single enzyme complex, a hydrolytic rate constant which is independent of substrate length, and a ratelimiting hydrolytic step. Recent kinetic studies with GA contradict these assumptions. Here we reevaluate the substrate binding of GA studying the pre-steady-state kinetics with glucose, which is reported here for the first time, and maltose. The association equilibrium constants for glucose and maltose interactions with wild- type and Trp120-->Phe GA from Aspergillus awamori in water and D2O buffers were obtained. Kinetic results indicate that a single glucose molecule binds to GA weakly by a single-step mechanism, E + G1EG1, under the conditions studied. Similar fluorescence intensities of the GA-glucose and GA-maltose complexes, the high tryptophan concentration around subsite 1, crystal structures of various inhibitor complexes, pre-steady-state and steady-state modeling, feasibility of condensation reactions, and other evidence strongly suggest that glucose binds at subsite 1. These results conflict with the high subsite 2 and low subsite 1 affinities obtained using Hiromi's model. Using the substrate association constants for glucose and maltose obtained by pre-steady- state kinetics, the affinity of α-glucose for subsite 1 is shown to be substantially higher than the apparent affinity of glucose for subsite 2. We propose a GA catalytic mechanism whereby substrate binding is initiated by subsite 1 interactions with the nonreducing end of the oligosaccharide substrate, minimizing nonproductive substrate binding. Through conformational changes, entropic contributions, and increased local concentration, subsite 2 subsequently has enhanced affinity for the second covalently linked glucosyl residue.
Enzyme, activity Kinetic

"Evidence For A Glutathionyl-enzyme Intermediate In The Amidase Activity Of The Bifunctional Glutathionylspermidine Synthetase/amidase From Escherichia Coli"
Biochemistry 1997 Volume 36, Issue 48 Pages 14930-14938
Chun-Hung Lin, David S. Kwon, J. Martin Bollinger, Jr., and Christopher T. Walsh

Abstract: Glutathionylspermidine (Gsp) is a metabolite common to Escherichia coli and protozoal parasites of the Trypanosoma family. Though its role in E. coli is unknown, Gsp is known to be an intermediate in the biosynthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), a metabolite unique to trypanosomatids that may allow the parasites to overcome oxidative stresses induced by host defense mechanisms. The bifunctional Gsp-synthetase/amidase from E. coli catalyzes both amide bond formation and breakdown between the N1-amine of spermidine [N-(3-aminopropyl)-1,4-diaminobutane] and the glycine carboxylate of glutathione (γ-Glu-Cys-Gly), with net hydrolysis of ATP [Bollinger et al. (1995) J. Biol. Chem. 270 (23), 14031-14041]. Synthetase and amidase activities reside in separate domains of the protein, and liberation of the amidase domain from the synthetase domain activates the amidase activity as much as 70-fold in kcat/K(m) for a chromogenic substrate γ-Glu-Ala-Gly-pNA [Kwon et al., (1997) J. Biol. Chem. 272 (4), 2429-2436]. When substrates for the Gsp-synthetase activity are present (GSH, ATP-Mg2+), Gsp-amidase is highly activated (15-fold). We provide kinetic and mutagenesis evidence suggesting that the amidase operates by a nucleophilic attack mechanism involving cysteine as the catalytic nucleophile. Stopped-flow studies on the 25 kDa Gsp-amidase fragment and the 70 kDa full-length Gsp-synthetase/amidase with γ- Glu-Ala-Gly-ONp demonstrate burst kinetics characteristic of a covalent acyl-enzyme intermediate. Studies using various group-specific protease inhibitors, such as iodoacetamide, suggest an active-site cysteine or histidine as being relevant to amidase activity, and site-directed mutagenesis indicates that Cys-59 is essential for amidase activity.
Enzyme, activity Bacteria Fermentation broth Stopped-flow Kinetic

"Estimation Of The Distance Change Between Cysteine-457 And The Nucleotide Binding Site When Sodium Pump Changes Conformation From E1 To E2 By Fluorescence Energy Transfer Measurements"
Biochemistry 1996 Volume 35, Issue 25 Pages 8419-8428
Shwu-Hwa Lin and Larry D. Faller

Abstract: The first indication of the size of a conformational change implicated in ion transport by sodium pump has been obtained by measuring the change in efficiency of fluorescence energy transfer between two specific locations on the α-subunit. The donor (5'- (iodoacetamido)fluorescein) attaches covalently to cysteine-457, and the acceptor (2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate) binds reversibly to the active site. The acceptor binds nearly 2 orders of magnitude tighter to the Na+ than to the K+ conformation of the enzyme and quenches donor fluorescence more efficiently in the Na+ than in the K+ conformation. The estimated distance between donor and acceptor, assuming random orientation of their emission and absorption dipoles, increases 2.9±0.6 A when the enzyme changes from the Na+ to the K+ conformation. Stopped-flow measurements of the change in fluorescence energy transfer efficiency with time when the doubly- labeled pump is mixed with Na+ or K+ demonstrate that the donor/acceptor pair reports the change between the E1 and E2 conformations of unphosphorylated enzyme. The observed first-order rate constant for the change in energy transfer efficiency depends sigmoidally on [K+] and inversely on [Na+], and both rate and amplitude data for the change in energy transfer efficiency can be fit with the same values of the rate and ion-dissociation constants as published data for the conformational change between E1 and E2 obtained by singly labeling the enzyme with fluorophores that report changes in protein microenvironment. The prerequisite for successfully measuring the distance change and equating the protein rearrangement with a step in the catalysis-transport cycle is that the donor by itself does not report the conformational change.
Protein, folding Fluorescence Stopped-flow Kinetic Titrations

"Spectral And Kinetic Studies On The Activation Of Soluble Guanylate Cyclase By Nitric Oxide"
Biochemistry 1996 Volume 35, Issue 4 Pages 1093-1099
James R. Stone and and Michael A. Marletta

Abstract: The soluble form of guanylate cyclase (sGC) is the only definitive receptor for the signaling agent nitric oxide (.NO). The enzyme is a heterodimer of homologous subunits in which each subunit binds 1 equivalent of 5-coordinate high-spin heme. .NO increases the Vmax of sGC up to 400- fold and has previously been shown to bind to the heme to form a 5- coordinate complex. Using stopped-flow spectrophotometry, it is demonstrated that the binding of .NO to the heme of sGC is a complex process. .NO first binds to the heme to form a 6-coordinate nitrosyl complex, which then converts to a 5-coordinate nitrosyl complex through one of two ways. For 28±4% of the heme, the 6-coordinate nitrosyl complex rapidly (approximately 20 s-1) converts to the 5-coordinate complex. For the remaining 72±4% of the heme, the conversion of the 6-coordinate nitrosyl complex to a 5-coordinate nitrosyl complex is slow (0.1-1.0 s-1) and is dependent upon the interaction of .NO with an unidentified non-heme site on the protein. The heme (200 nM) was completely converted to the 5-coordinate state with as little as 500 nM .NO, and the equilibriumibrium dissociation constant of .NO for activating the enzyme was determined to be < or = 250 nM. Gel-filtration analysis indicates that the binding of .NO to the heme has no effect on the native molecular mass of the protein. Correlation of electronic absorption spectra with activity measurements indicates that the 5- coordinate nitrosyl form of the enzyme is activated relative to the resting 5-coordinate ferrous form of the enzyme.
Enzyme, activity Spectrophotometry Kinetic Stopped-flow

"Spectroscopic, Calorimetric, And Kinetic Demonstration Of Conformational Adaptation In Peptide-antibody Recognition"
Biochemistry 1995 Volume 34, Issue 50 Pages 16509-16518
Lukas Leder, Christine Berger, Susanne Bornhauser, Hans Wendt, Friederike Ackermann, Ilian Jelesarov, and Hans Rudolf Bosshard

Abstract: Little is known about the extent to which protein flexibility contributes to antigen-antibody recognition and cross-reactivity. Using short coil peptides (leucine zippers) as model antigens, we demonstrate that a monoclonal antibody can force a noncognate peptide into a conformation that is similar to the conformation of the cognate peptide against which the monoclonal antibody is directed. Monoclonal antibodies 29AB and 13AD were raised against the 29-residue peptide LZ (Ac-EYEALEKKLAALEAKLQALEKKLEALEHG-amide) that forms a very stable coiled coil. The two antibodies cross-reacted strongly with the random coil analogue LZ(7P14P) that contains Lys-->Pro and Ala-->Pro substitutions in positions 7 and 14, respectively. The antibody-bound peptide LZ(7P14P) adopted an altered conformation that possibly was coiled coil-like, as shown by CD difference spectroscopy and fluorescence quenching experiments on coumarin-labeled peptides. Isothermal titration calorimetry revealed that the cross-reaction of antibodies 13AD and 29AB with the random coil peptide LZ(7P14P) exhibited a large unfavorable entropy. This, however, was strongly compensated by a more favorable enthalpy, resulting in only a small difference between the association constants for peptide LZ and LZ(7P14P), respectively. To investigate the opposite type of cross- reaction, monoclonal antibody 42PF was raised against the random coil peptide LZ(7P14P). 42PF cross-reacted with coiled coil peptide LZ by forcing it to dissociate into single chains. Enthalpy/entropy compensation again enabled the cross-reaction, which now was entropically favored and enthalpically disfavored. The rate of reaction of antibody 42PF with peptide LZ was controlled by the rate of dissociation of LZ into single chains. This observation, as well as the generally much slower reaction rate with the noncognate peptides, indicated that the cross-reactivity occurred because the antibody selected the conformer of the antigen that binds the strongest, a mechanism we call 'induced fit by conformational selection.' (FIA was used!)
Protein, structure Mass spectrometry Calorimetry Fluorescence Kinetic

"The Ferrous Heme Of Soluble Guanylate Cyclase: Formation Of Hexacoordinate Complexes With Carbon Monoxide And Nitrosomethane"
Biochemistry 1995 Volume 34, Issue 50 Pages 16397-16403
James R. Stone and Michael A. Marletta

Abstract: The soluble form of guanylate cyclase (sGC) is the only definitive receptor for the signaling agent nitric oxide (NO). The enzyme is a homologous heterodimer in which each subunit binds 1 equivalent of five-coordinate high-spin heme. NO increases the Vmax of sGC up to 400-fold, probably by binding directly to the heme. Carbon monoxide (CO) forms a six-coordinate complex with the heme and weakly activates the enzyme. Using stopped-flow spectrophotometry, the on-rate and off-rate for the binding of CO to the heme have been determined to be 3.58±0.15 x 10^4 M-1 s-1 and 3.5±0.5 s-1, respectively, at 10°C. The equilibrium dissociation constant (Kd) has been independently determined to be 97±9 µM. Comparison of this Kd with that calculated from the rate constants indicates that the binding of CO to sGC is a simple one-step process, in which the off-rate of CO from the hexacoordinate complex is much faster than typically found in hemoproteins. The Kd of CO for activating the enzyme was also determined and compared to that for binding to the heme. Nitrosomethane forms irreversible complexes with typical ferrous hemoproteins but was observed to bind reversibly to the heme in sGC, with an off-rate > or = 7.6±0.2 x 10^-3 s-1. In general, the ferrous heme in sGC has a low affinity for ligands that form six-coordinate complexes due primarily to fast ligand off-rates.
Carbon dioxide Spectrophotometry Immobilized enzyme Stopped-flow Complexation

"Electron Transfer Reactions Between Aromatic Amine Dehydrogenase And Azurin"
Biochemistry 1995 Volume 34, Issue 38 Pages 12249-12254
Young-Lan Hyun and Victor L. Davidson

Abstract: Binding and electron transfer reactions between the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH), and the type I copper protein azurin have been characterized. In steady- state kinetic assays using azurin as an electron acceptor, it was observed that the apparent Km for azurin decreased with increasing ionic strength. These results are the opposite of what was observed for the reaction between the TTQ enzyme methylamine dehydrogenase (MADH) and amicyanin, despite the fact that in both cases the pairs of redox proteins are each acidic proteins. It was further demonstrated that azurin does not function as an effective electron acceptor for MADH, and that amicyanin does not function as an effective electron acceptor for AADH. Thus, while the two TTQ enzymes each use type I copper proteins as physiologic electron acceptors, there is a strong specificity for which copper protein serves as a redox partner. The kinetic parameters for the electron transfer reactions from reduced AADH to oxidized azurin were determined by stopped-flow spectroscopy. Different results were obtained depending upon whether AADH was reduced chemically with dithionite or with the substrate tyramine. The values for the limiting first-order apparent electron transfer rate constant (kET) at 30°C were 4 and 102 s-1, respectively. Kinetically determined values of Kd also differed by a factor of 2.4. These data suggest that the incorporation of the substrate-derived amino group into the reduced TTQ of AADH significantly increases the apparent kET. The interaction between AADH and azurin was also quantitated using an ultrafiltration binding assay. This yielded a Kd of 300 µM for the AADH--azurin complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Azurin Amine dehydrogenase Spectrophotometry Kinetic Stopped-flow

"Rapid Plasmenylethanolamine-selective Fusion Of Membrane Bilayers Catalyzed By An Isoform Of Glyceraldehyde-3-phosphate Dehydrogenase: Discrimination Between Glycolytic And Fusogenic Roles Of Individual Isoforms"
Biochemistry 1995 Volume 34, Issue 38 Pages 12193-12203
Paul E. Glaser and Richard W. Gross

Abstract: Recently we demonstrated that the unique stereoelectronic relationships inherent in the structure of plasmenylethanolamine facilitate membrane fusion, and we postulated the existence of a membrane fusion protein which could exploit the propensity of plasmenylethanolamine molecular species to adapt an inverted hexagonal phase [Glaser and Gross (1994) Biochemistry 33, 5805-5812]. We now report a cryptic membrane fusion activity in rabbit brain cytosol, which requires separation from an endogenous inhibitor to express its activity, and demonstrate that vesicle fusion catalyzed by this protein is highly selective for membrane vesicles containing plasmenylethanolamine. The cytosolic protein catalyzing membrane fusion activity was purified to apparent homogeneity by sequential column chromatographies, revealing a single 38-kDa protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Automated Edman degradation demonstrated that the purified protein is an isoform of glyceraldehyde- 3-phosphate dehydrogenase (GAPDH), which was confirmed by Western blot analysis utilizing polyclonal antibodies and by solution-state inactivation of membrane fusion activity by a monoclonal antibody directed against GAPDH. Both GTP-affinity and Mono Q chromatographies resolved GAPDH isoforms that catalyzed dehydrogenase activity from the GAPDH isoform that catalyzed membrane fusion activity. The purified fusion protein was calcium-independent, resistant to treatment with N- ethylmaleimide, and possessed an obligatory requirement for plasmenylethanolamine and cholesterol. High-resolution stopped-flow kinetic analysis of plasmenylethanolamine-facilitated membrane fusion demonstrated that one tetramer of the GAPDH isoform catalyzed one fusion event between two vesicles containing plasmenylethanolamine every millisecond (on average). Collectively, these results constitute the first description of a protein which can catalyze the fusion of vesicles at a rate which satisfies the mathematical constraints imposed by the observed rates of fusion of synaptic vesicles with the presynaptic membrane in vivo.
Plasmenylethanolamine Brain Stopped-flow

"Diffusion-limited Interaction Between Unfolded Polypeptides And The Escherichia Coli Chaperone SecB"
Biochemistry 1995 Volume 34, Issue 31 Pages 10078-10085
Peter Fekkes, Tanneke den Blaauwen, and Arnold J. M. Driessen

Abstract: SecB is a chaperone dedicated to protein translocation in Escherichia coli. SecB binds to a subset of precursor proteins, and targets them in a translocation-competent state to the SecA subunit of the translocase. The nature and kinetics of the interaction of SecB with polypeptides were studied by spectroscopic techniques using the reduced form of bovine pancreatic trypsin inhibitor (BPTI) as a model substrate. Binding of SecB to BPTI resulted in an increase in the fluorescence of the surface-exposed tryptophan residue 36 of SecB. SecB reversibly binds BPTI in stoichiometric amounts. Labeling of BPTI with the fluoriphore acrylodan allowed the analysis of the binding reaction at nanomolar concentrations. High-affinity binding (KD of 5.4 nM) of labeled BPTI to SecB resulted in a blue shift of the acrylodan emission maximum and an increase in the fluorescence quantum yield, suggesting that BPTI binds in an apolar environment. Stopped-flow acquisition of rate constants of complex formation between SecB and BPTI yielded a second-order binding rate constant of 5 x 10(9) M-1 s-1, and a dissociation rate constant of 48 s-1. These data demonstrate that in vitro, the association of SecB with polypeptide substrates is limited by the rate of collision. In vivo, SecB binding is selective, and predominantly occurs with nascent polypeptides. Since these chains are not expected to fold into stable structures, SecB association may be governed by 'more or less' specific interactions and be limited by the rate of chain elongation rather than the rate of folding.
Enzyme, SecA Fluorescence Kinetic Stopped-flow

"Oxidation Of Yeast Iso-1 Ferrocytochrome C By Yeast Cytochrome C Peroxidase Compounds I And II. Dependence Upon Ionic Strength"
Biochemistry 1995 Volume 34, Issue 31 Pages 9991-9999
Andrea L. Matthis, Lidia B. Vitello, and James E. Erman

Abstract: The reduction of cytochrome c peroxidase compound I by excess yeast iso- 1 ferrocytochrome c is biphasic. Two pseudo-first-order rate constants can be measured by stopped-flow techniques. The fastest rate process is the reduction of cytochrome c peroxidase compound I to compound II, and the slower process is the reduction of II to the native enzyme. The yeast iso-1 ferrocytochrome c concentration dependence of the reduction of cytochrome c peroxidase compound I to compound II is consistent with a mechanism involving two binding sites for cytochrome c on cytochrome c peroxidase. Electron transfer from cytochrome c bound at the high- affinity binding site to the Fe(IV) site in cytochrome c peroxidase compound I is dependent upon ionic strength, increasing from 15±6 to 2000±100 s-1 over the ionic strength range 0.01-0.20 M. The reduction rate of the Fe(IV) site in the 2:1 yeast iso-1 ferrocytochrome c/cytochrome c peroxidase compound I complex is essentially independent of ionic strength with a value of 3800±300 s-1. The Fe(IV) site in cytochrome c peroxidase compound I is preferentially reduced by yeast ferrocytochrome c between 0.01 and 0.20 M ionic strength while the Trp-191 radical is preferentially reduced above 0.30 M ionic strength. The association rate constant for the binding of yeast iso-1 ferrocytochrome c to cytochrome c peroxidase compound I can be evaluated and varies from a remarkable 1 x 10(10) M-1 s-1 at 0.01 M ionic strength to 1.2 x 10(5) M-1 s-1 at 1.0 M ionic strength. Between 0.01 and 0.20 M ionic strength, the reduction of cytochrome c peroxidase compound II to the native enzyme is anomalous. The reaction is independent of the cytochrome c concentration and directly proportional to the initial cytochrome c peroxidase compound I concentration.
Enzyme, cytochrome c peroxidase Fermentation broth Rate constants Stopped-flow Ionic strength

"Kinetic Evidence For Ternary Complex Formation And Allosteric Interactions In Chloride And Stilbenedisulfonate Binding To Band 3"
Biochemistry 1994 Volume 33, Issue 39 Pages 11909-11916
James M. Salhany, Renee L. Sloan, Karen A. Cordes, and Lawrence M. Schopfer

Abstract: The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4'- dibenzamidostilbene-2,2'-dibenzamidostilbene-2,2'-disulfonate) and H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) by more than 10-fold at constant ionic strength. The acceleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of KI-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.
Stilbenedisulfonate Ion exchange Fluorescence Kinetic

"Fluorescence Studies Of Phosphatidylcholine Micelle Mixing: Relevance To Phospholipase Kinetics"
Biochemistry 1994 Volume 33, Issue 38 Pages 11608-11617
Christine E. Soltys and Mary F. Roberts

Abstract: Two fluorescent micellar phospholipid probes (1-hexanoyl-2-(1- pyrenebutyroyl)phosphatidylcholine and 1-octanoyl-2-(1- pyrenebutyroyl)phosphatidylcholine) have been synthesized, characterized, and used to monitor the dynamics of lipid/amphiphile exchange in a variety of detergents and phospholipid micelles using both steady-state and stopped-flow fluorescence techniques. The ratio of the pyrene monomer to excimer band is a good indicator of the extent of lipid mixing at equilibrium. Following the time dependence of increase in the monomer band with stopped-flow methodology provides a rate constant for this exchange process (most systems were well fit with a single exponential). Short-chain pyrene-labeled phosphatidylcholine mixing with Triton X-100 micelles is extremely fast and follows a concentration dependence indicative of the importance of micelle collisions for the exchange process. Submicellar amounts of Triton have no effect on the fluorescent dynamics of the probe molecule. Other detergents such as β-octyl glucoside and deoxycholate are also effective at higher concentrations, although significant differences exist in the extent of probe mixing. Short- chain diacylphosphatidylcholine and lysophosphatidylcholine mixing rates are moderately fast with mixing times that decrease as the hydrophobicity/chain length of the diluent matrix increases. The rate constants for lipid exchange can be compared to turnover rates of several phospholipases in these assay systems. Anomalous mixing behavior of unusual micelle forming lipids [bolaforms and omega- carboxylate phosphatidylcholines [Lewis, K. A., Bian, J., Sweeny, A., and Roberts, M. F. (1994) Biochemistry 29, 9962-9970] and polymerizable phosphatidylcholines [Soltys, C. E., Bian, J., and Roberts, M. F. (1993) Biochemistry 32, 9545-9551] is particularly helpful in understanding kinetics of water-soluble phospholipases on these systems.
Phospholipids Fluorescence Micelle Triton X Stopped-flow Kinetic Surfactant

"Kinetic And Equilibrium Studies Of Porphyrin Interactions With Unilamellar Lipidic Vesicles"
Biochemistry 1994 Volume 33, Issue 32 Pages 9447-9459
Katerina Kuzelova and Daniel Brault

Abstract: The interaction of deuteroporphyrin with dimyristoylphosphatidylcholine unilamellar vesicles of various sizes (ranging from 38 to 222 nm) has been studied using a stopped-flow with fluorescence detection. Beside the kinetics of porphyrin incorporation into vesicles, the transfer of porphyrin from vesicles to human serum albumin has been investigated both experimentally and theoretically. The effects of both vesicle and albumin concentrations indicate that the transfer proceeds through the aqueous phase. It is governed by the rate of incorporation of porphyrin into the outer vesicle hemileaflet (kon), by the exit to the bulk aqueous medium (koff), and by the association (kas) and dissociation (kdis) constants relative to albumin. In both systems studied, a slower transbilayer flip-flop accounts for the biphasic character of the kinetics. This model is strongly supported by the effects of vesicle size, temperature, and cholesterol. The dependence of kon on the vesicle size indicates that the incorporation is diffusion controlled. The constant koff is found to be closely coupled to the phase state of the bilayer. The transbilayer flip-flop rate constant is approximately the same in both directions (approximately 0.4 s-1 at 32°C and pH 7.4). It is strongly affected by the presence of cholesterol in vesicles and by the temperature, with a sharp enhancement around the phase transition. With the exception of very small vesicles obtained by sonication, no influence of the vesicle size on the flip-flop rate was observed. An accelerating effect of tetrahydrofuran, used to improve the solubility of porphyrin, has been noted. Steady-state measurements and kinetics results were in excellent agreement. The interest of systems involving albumin as a scavenger to extract important rate constants, is emphasized.
Porphyrin Serum Human Fluorescence Kinetic Liposomes Stopped-flow

"Role Of Tyrosine 143 In Lactate Dehydrogenation By Flavocytochrome B2. Primary Kinetic Isotope Effect Studies With A Phenylalanine Mutant"
Biochemistry 1994 Volume 33, Issue 3 Pages 798-806
Nathalie Rouviere-Fourmy, Chantal Capeillere-Blandin, and Florence Lederer

Abstract: Flavocytochrome b2 catalyzes the oxidation of lactate at the expense of cytochrome c. After flavin (FMN) reduction by the substrate, reducing equivalents are transferred one by one to heme b2, and from there on to cytochrome c. The crystal structure of the enzyme is known at 2.4-A resolution, and specific roles in catalysis have been assigned to active side chains. Tyr143 in particular, located at the interface between the flavodehydrogenase moiety and the heme-binding domain, was thought to take part in substrate binding, as well as to orient the heme-binding domain for efficient electron transfer. A first study of the properties of a Tyr143Phe mutant showed that the major effect of the mutation was to decrease the rate of electron transfer from flavin to heme [Miles, C.S., Rouviere-Fourmy, N., Lederer, F., Mathews, F.S., Reid, G.A., Black, M.T., and Chapman, S.K. (1992) Biochem. J. 285, 187- 192]. In the present paper, we focus on the effect of the mutation on catalysis of lactate dehydrogenation. We report the deuterium kinetic isotope effects on flavin reduction as measured with stopped-flow methods and on cytochrome c reduction in the steady-state using L-[2- 2H]lactate. For the wild-type enzyme, isotope effects on FMN reduction, D(kredF) and D(kredF)/Km), were 7.2±0.9 and 4.2±1.3, respectively, and for the Y143F mutant values of 4.4±0.5 and 3.9±1.1 were obtained. Calculations, from deuterium isotope effects, of substrate Kd values, combined with knowledge of kcat/Km values, lead to the conclusion that Tyr143 does stabilize the Michaelis complex by hydrogen bonding to a substrate carboxylate, as was postulated; but the mutation does not destabilize the transition state more than the Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Tyrosine Biological Catalysis Stopped-flow Kinetic

"Reconstitution Of Purified GABAA Receptors: Ligand Binding And Chloride Transporting Properties"
Biochemistry 1994 Volume 33, Issue 3 Pages 755-763
Susan M. J. Dunn and Rick P. Thuynsma

Abstract: GABAA receptors have been solubilized from bovine brain membranes and, following purification by benzodiazepine affinity chromatography, have been reconstituted into phospholipid vesicles. Reconstituted vesicles were about 120 nm in diameter, and, on average, each vesicle contained fewer than one GABAA receptor which was reconstituted in an outside-out orientation. These preparations have been used in parallel studies of radiolabeled ligand binding and chloride flux, the latter being measured by following the fluorescence changes of a chloride-sensitive probe which was trapped within the vesicles at the time of reconstitution. The benzodiazepine [3H]flunitrazepam binds to an apparently homogeneous population of sites in these preparations (Kd of 5 nM) whereas the GABA analogue [3H]muscimol binds to both high- and low-affinity sites (KdS of 10 nM and 0.27 µM). Muscimol stimulated chloride flux with an EC50 of 0.2 µM and, at similar concentrations (EC50 = 0.16 µM), potentiated [3H]flunitrazepam binding, suggesting that occupancy of the low-affinity sites may be important for these effects. Diazepam shifted the dose-response curve for muscimol- stimulated flux to about 4-fold lower concentrations without affecting the maximum response. Diazepam did not, however, alter the equilibrium binding of [3H]muscimol. The purified receptor showed densensitization since flux responses were abolished by prior exposure to muscimol. The competitive antagonist bicuculline and the channel blocker picrotoxin completely inhibited ion flux mediated by 3 µM muscimol with EC50 values of 5.3 and 2.5 µM, respectively. These results are discussed in terms of possible mechanisms for activation, inhibition, and modulation of GABAA receptors.
Chloride Flunitrazepam Cow Serum LC

"Demonstration Of A Two-step Reaction Mechanism For The Inhibition Of Heparin-bound Neutrophil Elastase By α 1-proteinase Inhibitor"
Biochemistry 1993 Volume 32, Issue 35 Pages 9230-9235
Bernard Faller, Martine Cadene, and Joseph G. Bieth

Abstract: Heparin decreases the rate of inhibition of neutrophil elastase by α 1-proteinase inhibitor as a result of its strong binding to the enzyme. Here, we used the slow-binding kinetic approach to decide whether the enzyme-inhibitor interaction proceeds via a two-step mechanism and to identify the step that is affected by heparin. The inhibition kinetics was assessed under pseudo-first-order conditions using conventional or stopped-flow spectrophotometry. In the absence of heparin, the pseudo-first-order rate constant of inhibition increased linearly with the inhibitor concentration indicating that within the experimental concentration range (EI with kass = 10(7) M-1 s-1) or to a two-step reaction (E+I KiEI k2-->EI with Ki > 0.4 µM and k2 > 4 s-1). In the presence of heparin, the rate constant of inhibition varied hyperbolically with the inhibitor concentration, indicating that the inhibition is a two-step process with Ki = 80 nM and K2 = 0.15 s-1. Thus, heparin has two opposite effects on the elastase + α 1- proteinase inhibitor interaction: it favors the association by decreasing Ki but impairs it by decreasing k2. This rationalizes the previously demonstrated rate-depressing effect of the sulfated polymer. Heparin does not significantly alter the stability of the irreversible elastase-α 1-proteinase inhibitor complex.
Enzyme, inhibition Spectrophotometry Stopped-flow Kinetic

"Lifetime Of The Histone Octamer Studied By Continuous-flow Quasi-elastic Light-scattering - Test Of A Model For Nucleosome Transcription"
Biochemistry 1993 Volume 32, Issue 30 Pages 7824-7831
H. P. Feng, Dale S. Scherl, and J. Widom

Abstract: An instrument for continuous-flow quasielastic light scattering is described that allows the translational diffusion coefficient of macromolecules to be determined as a function of time after the initiation of some time-dependent process by mixing. Control experiments are carried out using the proteins lysozyme and BSA to verify that flow of the solution does not lead to erroneous results. The instrument is used to determine the lifetime of the histone octamer. A solution of octamer that is artificially stabilized in 2.0 M NaCl is rapidly diluted to physiological ionic strength, and the Stokes diameter is determined as a function of the time, At, after mixing. We find that the octamers dissociate into their component H2AH2B heterodimers and H32H42 tetramers on a time scale that is faster than the earliest time point for which data were obtained, 1 s after mixing. This result argues against a simple mechanism for the progression of RNA or DNA polymerase through chromatin.

"Reaction Of Indole And Analogs With Amino Acid Complexes Of Escherichia Coli Tryptophan Indole-lyase: Detection Of A New Intermediate By Rapid Scanning Stopped-flow Spectrophotometry"
Biochemistry 1991 Volume 30, Issue 24 Pages 5927-5934
Robert S. Phillips

Abstract: The effects of indole and analogues on the reaction of Escherichia coli tryptophan indole-lyase (tryptophanase) with amino acid substrates and quasisubstrates have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Indole binds rapidly (within the dead time of the stopped-flow instrument) to both the external aldimine and quinonoid complexes with L-alanine, and the absorbance of the quinonoid intermediate decreases in a subsequent slow relaxation. Indoline binds preferentially to the external aldimine complex with L-alanine, while benzimidazole binds selectively to the quinonoid complex of L-alanine. Indole and indoline do not significantly affect the spectrum of the quinonoid intermediates formed in the reaction of the enzyme with S-alkyl-L-cysteines, but benzimidazole causes a rapid decrease in the quinonoid peak at 5 12 nm and the appearance of a new peak at 345 nm. Benzimidazole also causes a rapid decrease in the quinonoid peak at 505 nm formed in the reaction with L-tryptophan and the appearance of a new absorbance peak at 345 nm. Furthermore, addition of benzimidazole to solutions of enzyme, potassium pyruvate, and ammonium chloride results in the formation of a similar absorption peak at 340 nm. This complex reacts rapidly with indole to form a quinonoid intermediate very similar to that formed from L-tryptophan. This new intermediate is formed faster than catalytic turnover (kat = 6.8 s-I) and may be an a-aminoacrylate intermediate bound as a gem-diamine.
Spectrophotometry Enzyme Kinetic Stopped-flow