Active Site Thiol Group Research Articles

Alpha-thrombin-catalyzed activation of human platelet factor XIII: relationship between proteolysis and factor XIIIa activity.

The kinetics of activation of platelet factor XIII, an a-subunit dimer, were characterized by determining rate constants for activation peptide (AP) release, generation of activity, and exposure of the active-site thiol group. The specificity constant (kappacat/Km) for alpha-thrombin-catalyzed AP release, 1.2 x 10(5) M-1s-1, was found to be similar to that for AP release from the tetramer plasma factor XIII (a2b2) [Janus, T.J., Lewis, S. D., Lorand, L., & Shafer, J. A. (1983) Biochemistry 22, 6269-6272], implying that the b subunits of plasma factor XIII do not hinder alpha-thrombin-catalyzed cleavage of AP from the a subunit. Platelet factor XIIIa activity was generated at a rate approximately twice the rate of AP release. This difference in rates was shown to be consistent with a reaction pathway for activation of platelet factor XIII wherein full factor XIIIa activity is generated when one AP is removed from the dimeric zymogen so that removal of the second AP has no detectable effect on catalytic activity. In accord with this conclusion, the rate constant for exposure of the active-site thiol group, as measured by the incorporation of [1-14C]-iodoacetamide, was about twice that observed for the removal of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical modification of the RTEM-1 thiol beta-lactamase by thiol-selective reagents: evidence for activation of the primary nucleophile of the beta-lactamase active site by adjacent functional groups.

The RTEM-1 thiol beta-lactamase (Sigal, I.S., Harwood, B.G., Arentzen, R., Proc. Natl. Acad. Sci. U.S.A. 79:7157-7160, 1982) is inactivated by thiol-selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4'-dipyridyldisulfide, which modify the active site thiol group. The pH-rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form. The pKa of the active site thiol is therefore shown by the data to be below 4.0. This low pKa is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion. The low nucleophilicity of the thiolate in both EH2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol beta-lactamase is stabilized by two hydrogen-bond donors. One of these, of pKa greater than 9.0, is suggested to be the conserved and essential Lys-73 ammonium group, while the identity of the other group, of pKa around 6.7, is less clear, but may be the conserved Glu-166 carboxylic acid. beta-Lactamase activity is associated with the EH2 form, and thus the beta-lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol beta-lactamase) and two acidic (hydrogen-bond donor) groups (one of which is likely to be the above-mentioned lysine ammonium group).

Inactivation of gamma-glutamylcysteine synthetase, but not of glutamine synthetase, by S-sulfocysteine and S-sulfohomocysteine.

The reactions catalyzed by gamma-glutamylcysteine synthetase and glutamine synthetase are thought to proceed via enzyme-bound gamma-glutamyl phosphate intermediates. We investigated the possibility that S-sulfocysteine and S-sulfohomocysteine might act as analogs of gamma-glutamyl phosphate or of the associated putative tetrahedral intermediates. The D- and L-enantiomers of S-sulfocysteine and S-sulfohomocysteine were found to rapidly inactivate rat kidney gamma-glutamylcysteine synthetase but to be reversible inhibitors of sheep brain glutamine synthetase. Inactivation of gamma-glutamylcysteine synthetase does not require ATP and is associated with noncovalent binding of close to 1 mol of inactivator/mol of enzyme. The findings indicate that the S-sulfo amino acids are transition-state analogs, and that binding of S-sulfo amino acid to the enzyme induces formation of a very stable enzyme-inactivator complex. The data suggest that stabilization of the enzyme-inactivator complex results from interactions involving the sulfenyl sulfur atom of the S-sulfo amino acid and the active site thiol group of the enzyme.

Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.

Determination of a low pK for histidine-159 in the S-methylthio derivative of papain by proton nuclear magnetic resonance spectroscopy.

Proton NMR spectroscopy was used to study the ionization behavior of His-159 in a derivative of papain (papain-S-SCH3). In this catalytically inactive derivative of papain, the active-site thiol group of Cys-25 is S-methyl-thiolated so that it cannot form a thiolate anion. The pH dependence of the chemical shift of the C epsilon 1 H resonance of His-159 indicated a pK of 3.45 +/- 0.07 at 45 degrees C in 2H2O with no added ions other than those required for titration. In acetate buffers at an ionic strength of 0.05, the pK increased to 3.87 +/- 0.12. Conversion of papain-S-SCH3 to active papain at pH* 4.17 (at 45 degrees C and an ionic strength of 0.05) caused the position of the C epsilon 1 H resonance to change from a position indicative of partial protonation of His-159 to a position indicative of full protonation, consistent with the existence of an imidazolium-thiolate ion-pair interaction between His-159 and Cys-25 in the active enzyme.

P K perturbations of nitrophenol-containing reporter groups at the active site of papain

The p K perturbations exhibited by nitrophenol reporter groups linked to the active site thiol group in papain were compared in order to probe the pH dependence of the charge in the environment of the active site of this enzyme. Nitrophenol reporter groups were introduced at the active site of papain by reaction of the active site thiol group with 4-chloromercuri-2-nitrophenol ( 1a), 2-chloromercuri-4-nitrophenol ( 2a), and 2-chloromercuri-4,6-dinitrophenol ( 3a) to form papain derivatives 1c–3c. Spectrophotometric titrations of these derivatives of papain yielded p K values (25 °C, Γ 2 0.15) of 7.51, 7.99, and 3.93, for the reporter groups in 1c, 2c and 3c, respectively. Comparison of these p K values with those of the corresponding 2-mercaptoethanol derivatives 1b–3b indicates that the environment at the active site of papain caused upward perturbations of 0.35 and 0.96 unit in the p K's of the reporter groups in 1c and 2c, respectively, and a downward perturbation of 0.53 unit in the p K of the reporter group in 3c. These p K perturbations were compared to those determined previously for the nitrophenol reporter groups introduced at the active site of papain by reaction of papain with α-bromo-4-hydroxy-3-nitroacetophenone and 2-bromoacetamido-4-nitrophenol. Assuming that the phenolic function of geometrically similar reporter groups occupy similar positions at the active site of the enzyme, the observed p K perturbations exhibited by these five reporter groups indicate that the charge in the vicinity of the active site of papain (a) does not change between pH 5 and 8, and (b) becomes positive at pH values below pH 4–5. These conclusions are consistent with the view that the p K values of His-159 and Asp-158 at the active site of papain are both about 4 in forms of papain wherein the active site thiolate anion is neutralized by protonation or is blocked by chemical modification.

Inhibition of papain by N-acyl-aminoacetaldehydes and N-acyl-aminopropanones. Evidence for hemithioacetal formation by a cross-saturation technique in nuclear-magnetic resonance spectroscopy.

N-Acyl-aminoacetaldehydes are potent inhibitors of the proteolytic enzyme, papain. Although they exist predominantly in their hydrated form in aqueous solution only the aldehyde is an effective inhibitor. The binding constants for related amides and methyl ketones confirm that it is principally the lower steric requirement of the aldehyde rather than its increased electrophilicity which is responsible for its powerful inhibitor properties. Using nuclear magnetic resonance spectroscopy, evidence is provided for an N-acetyl-aminoacetaldehyde-papain complex. Using a cross-saturation technique evidence is also provided for a hemithioacetal, formed from the aldehyde and the active-site thiol group. Hemithioacetal formation has also been detected between N-benzoyl-aminoacetaldehyde and papain. This provides the first direct evidence for a tetrahedral adduct with papain and supports the proposed involvement of such intermediates in papain-catalysed hydrolyses.

Sulfhydryl groups in relation to the structure and catalytic activity of 2-OXO-4-hydroxyglutarate aldolase from bovine liver

Bovine liver 2-oxo-4-hydroxyglutarate aldolase (suggested name: 2-oxo-4hydroxyglutarate glyoxylate-lyase catalyzing the reaction: 2-oxo-4-hydroxyglutarate rlhar2 pyruvate + glyoxylate) contains eight to ten sulfhydryl groups as determined by titration of the enzyme with either 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs 2) or p-mercuribenzoate in the presence of 1% sodium dodecyl sulfate. In the absence of a denaturant, all of the cysteinyl residues react with p-mercuribenzoate whereas only four are accessible to titration with Nbs 2. No differences in -SH group reactivity can be detected during titration of the aldolase with p-mercuribenzoate. In contrast, two classes of sulfhydryls can be differentiated in the disulfide exchange reaction with Nbs 2 in the absence of a denaturant; one -SH group (Class I) reacts rapidly whereas three additional thiols (Class II) titrate at approx. 0.1 the rate of the Class I -SH residue. Both pyruvate and glyoxylate protect one of the three -SH residues in Class II from reaction with Nbs 2. Either substrate also prevents titration of one to two thiol groups by p-mercuribenzoate and decreases the rate of reaction of aldolase -SH groups with Nbs 2 in 8 M urea. These ligand-induced changes in -SH reactivity provide a sensitive indication that the enzyme exists in an altered conformational state in the presence of either of its cosubstrates. Titration of the enzyme with either Nbs 2 or p-mercuribenzoate results in a progressive loss of aldolase activity which is not proportional to the number of -SH groups modified. The enzyme retains 50% of the activity of the native enzyme when Class I and Class II thiols (i.e. four -SH groups total) are modified with Nbs 2; 15% residual activity is still observed following titration of all of the cysteinyl residues with p-mercuribenzoate. Pyruvate and glyoxylate provide partial protection against inactivation. It is concluded that inactivation of 2-oxo-4-hydroxyglutarate aldolase by Nbs 2 or p-mercuribenzoate is a consequence of alterations in protein structure which accompany modification of -SH groups. The data argue against the direct participation of an active-site thiol group in the catalytic mechanism of 2-oxo-4-hydroxyglutarate aldolase, be that aldol cleavage and condensation or β-decarboxylation.

Potentiometric determination of ionizations at the active site of papain.

The ionization behavior of groups at the active site of papain was determined from the pH dependence of the difference of proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by two independent methods. One method involved potentiometric measurements of the protons released and demethylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3 to 7.6 on deprotonation of His-159 at 29 degrees C/20.05. Similarly, the pK of His-159 shifts from 4.3 to 8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate that equilibrium constant for transfer of the proton from Cys-25 to His-159 is 8--12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.

Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.

1] Active site titration of cysteine proteases

Cysteine proteases catalyze the hydrolysis of carboxylic acid derivatives through a double-displacement pathway involving the general-acid general-base catalyzed formation and hydrolysis of an acyl-thiol intermediate. The great similarity of this mechanism to that displayed by serine proteases assures that the active site titration methods elaborated for serine proteases are also applicable without much modification to the determination of active site concentrations of sulfhydryl proteases. The presence of an active site thiol group as the acyl acceptor confers special properties to the sulfhydryl proteases, as compared to their serine analogs. Among amino acid side chain functions the SH group of cysteine is unique by its reactivity toward electrophilic reagents, oxidizing agents, and heavy metal ions. Since in most cysteine proteases the active-site thiol is the only sulfhydryl group on the protein molecule, the design of titrating agents is greatly facilitated by this unique reactivity. As an example, the use of chromophoric organomercurials could be singled out for the rapid and sensitive quantitation of sulfhydryl groups at specific sites. The chapter discusses the use of papain and bromelain as examples to illustrate some of the experimental approaches leading to the establishment of rate assays for cysteine proteases.

Active-site thiol groups of yeast hexokinase.

Research Article| December 01 1969 Active-site thiol groups of yeast hexokinase J G Jones J G Jones 1Department of Biochemistry, University College, Cathays Park, Cardiff Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1969) 115 (5): 41P. https://doi.org/10.1042/bj1150041Pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation J G Jones; Active-site thiol groups of yeast hexokinase. Biochem J 1 December 1969; 115 (5): 41P. doi: https://doi.org/10.1042/bj1150041Pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1969 The Biochemical Society1969 Article PDF first page preview Close Modal You do not currently have access to this content.