Second-order Rate Constants For Inactivation Research Articles

Reaction of phenylglyoxal with arginine groups in D-amino-acid oxidase from Rhodotorula gracilis.

D-Amino-acid oxidase from Rhodotorula gracilis was irreversibly inactivated by phenylglyoxal in a biphasic process. The fast phase was completed in less than 1 min. Its extent was linearly dependent on phenylglyoxal concentration and was not influenced by the presence of FAD or benzoate, a pseudo-substrate. The second phase of inactivation was due to a simple second-order reaction. The presence of FAD exerted only partial protection; the second-order rate constants of inactivation were 8.3 M-1 min-1 for holoprotein and 18.0 M-1 min-1 for apoprotein. The addition of benzoate completely protected against this second phase of inactivation. Efforts to isolate the enzyme modified at a single arginine residue at the end of the fast phase were unsuccessful, but analysis of the enzyme isolated at the end of the slow phase identified an arginine residue, protected by benzoate, that is highly conserved in all D-amino-acid oxidases and corresponds to Arg283 in the pig kidney enzyme. Modification of this residue is directly involved in the inactivation process during the slow phase. This arginine may represent the basic residue ion pairing with the carboxylate group of the substrate or the residue interacting with the flavin N1-C2 = O locus.

Inhibition of Streptomyces griseus metallo-endopeptidase II (SGMPII) by active-site-directed inhibitors.

Inactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 was studied kinetically. These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics. The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 were measured to be 0.12 and 8.9 M-1.s-1, respectively. The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin > SGMPII > Pseudomonas aeruginosa elastase. A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 in a competitive manner. Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme. These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme.

Novel N-peptidyl- O-acyl hydroxamates: selective inhibitors of cysteine proteinases

A series of N-peptidyl- O-acyl hydroxamates with a lysine in P 1 was synthesized and tested as inactivators of lysosomal cysteine proteinases (cathepsins S, L, B and H) and trypsin-like serine proteinases (trypsin, thrombin, plasmin t-PA). N-peptidyl- O-acyl hydroxamates were shown to be selective inhibitors of cysteine proteinases. With the exception of cathepsin H, the lysosomal cysteine proteinases were inactivated 2–5 orders of magnitude more rapidly than serine proteinases with a comparable primary substrate specificity. The highest second-order rate constants of inactivation for the cysteine proteinases are in the range of 10 5-10 6 M −1 s −1. The order of inhibitor specificity for the cysteine proteinases is comparable to the enzyme's substrate specificity.

N-Peptidyl- O-carbamoyl amino acid hydroxamates: Irreversible inhibitors for the study of the S 2′ specificity of cysteine proteinases

A series of new inhibitors for cysteine proteinases with the general structure Z-Phe-Gly-NHO-CO-Aa (Aa = amino acids) was synthesized and tested as inhibitors of papain-like enzymes (cathepsins S, L, B and papain). Like N-peptidyl- O-acyl hydroxamates the inhibitors inactivate cysteine proteinases by a sulfenamidation of the active site cysteine residue. The most effective inhibitors display second order-rate constants of inactivation in the range of 10 3–10 4 M −1·s −1. Since the structure of the N-peptidyl- O-carbamoyl amino acid hydroxamates allows the variation of the leaving group this class of inhibitors was used as a new tool for the evaluation of the S 2′ specificity of cysteine proteinases.

Iodo and diiodotyrosine epoxysuccinyl derivatives as selective inhibitors of cathepsin B

Eight new analogs of l- trans-epoxysuccinyl- l-leucylamido(3-methyl)butane (E-64-c) containing Phe, Tyr, Tyr(1) or Tyr(I 2) in place of Leu, were synthesized and tested as inhibitors of papain, bovine spleen cathepsin B, calpain I and II from porcine red cells and porcine kidney, respectively. By use of kinetic methods, the new E-64 analogs proved to irreversibly inactivate both papain and cathepsin B via reversible enzyme-inhibitor intermediates EI. Second-order rate constants for inactivation were in the range 3500-55 100 M −1s −1 for papain and 650–105 000 M −1s −1 for cathepsin B. For the inactivation of calpain I and II they ranged between 250 and 2000 M −1s −1 and were similar to those of the known E-64-c. The effectiveness of the amino acid contained in the inhibitors tested increased in the order Tyr(I) ≈ Tyr(I 2) < Tyr < Phe < Leu for papain and Phe < Tyr < Tyr(I) < Leu < Tyr(I 2) for cathepsin B inactivation. Replacement of the l with the d- trans-epoxysuccinyl unit caused a 10–100-fold decrease in inhibitor potencies.

N-Haloacetyl-amino-acid amides as active-site-directed inhibitors of papain and cathepsin B

A series of N-haloacetyl-amino-acid amides were synthesized and tested as models of cysteine-protease inhibitors. They irreversibly inactivated papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Apparent second-order rate constants of inactivation ranging from 65 to 16 700 M −1 s −1 were observed. Reactivity against papain, as compared to glutathione, was increased 16 400-fold for N-bromoacetyl-leucine isopentylamide and 25 700-fold for the corresponding iodoacetyl derivative; these increases are probably due to proximity effects. No inhibition of trypsin, chymotrypsin and porcine pancreatic elastase was observed. Haloacetamides represent an interesting class of easily synthesized, efficient, irreversible inhibitors of cysteine proteases, which have low non-specific alkylating properties.

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum

Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.

Nonpolar interactions in the maleimide inactivation of Haemophilus influenzaed-lactate dehydrogenase

A series of N-alkylmaleimides varying in chain length from N-ethyl up to and including N-heptyl, was shown to effectively inactive Haemophilus influenzae D-lactate dehydrogenase at pH 7.0 and 25°C in processes proposed to involve covalent modification of cysteine residues. The inactivation proceeded through an initial reversible binding of maleimides facilitated by nonpolar interactions with a hydrophobic region of the enzyme. Subsequent irreversible inactivation of the enzyme indicated the modification of a fast-reacting group leading to approx. 80% loss of enzyme activity followed by a second slower-reacting modification process. At saturating concentrations of maleimides, the second inactivation process exhibited a common pseudo-first-order rate constant of 0.6 min −1. The initial reversible binding of N-alkylmaleimides resulted in inhibition of the enzyme that was competitive with respect to NADH. Positive chain length effects were observed in the second-order rate constants for inactivation and in the 6-fold better binding of N-heptylmaleimide as compared to that for N-ethylmaleimide. It is suggested that the nonpolar interactions stabilizing the 1,4-dihydronicotinamide molety of the reduced coenzyme also facilitate the initial binding of N-alkylmaleimides.

Nonpolar interactions in the modification of an essential sulfhydryl of sorbitol dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides varying in chainlength from N-methyl- to N-octylmaleimide inclusive was shown to effectively inactivate sheep liver sorbitol dehydrogenase at pH 7.5 and 25 degrees C. The apparent second-order rate constants for inactivation increased with increasing chainlength of the N-alkylmaleimide used. Positive chainlength effects were also indicated by the Kd values for the N-ethyl and N-heptyl derivatives obtained from studies of the saturation kinetics observed for inactivation of the enzyme at high concentrations of these maleimides. The complete inactivation of sorbitol dehydrogenase was demonstrated to occur through the selective covalent modification of one cysteine residue per subunit of enzyme. The stoichiometry of enzyme inactivation was supported on the one hand by fluorescence titration with fluorescein mercuric acetate of the native and the inactivated enzyme, and, on the other hand, by the simultaneous inactivation of the enzyme with selective modification of one sulfhydryl per subunit by N-[p-(2-benzoxazolyl)phenyl]maleimide. Protection of the enzyme from N-alkylmaleimide inactivation was observed with the binding of NADH, whereas both NAD and sorbitol were ineffective as protecting ligands. Diazotized 3-aminopyridine adenine dinucleotide, in contrast to previous studies of this reagent with yeast alcohol dehydrogenase and rabbit muscle glycerophosphate dehydrogenase, did not function as a site-labeling reagent for sorbitol dehydrogenase.

Inactivation of chymotrypsin and human skin chymase: Kinetics of time-dependent inhibition in the presence of substrate

The reaction of chymase, a chymotrypric proteinase from human skin, and bovine pancreatic chymotrypsin with a number of time-dependent inhibitors has been studied. An integrated equation, relating product formation with rime, has been derived for the reaction of enzymes with time-dependent inhibitors in the presence of substrate. This is based on a two-step model in which a rapidly reversible, non-covalent complex (EI) is formed prior to a tighter, less readily reversible complex (EI)*). The equation depends on the simplifying assumption [I] ⪢ [E], but is applicable to reversible and irreversible slow-binding and tight-binding inhibitors whether or not they show saturation kineties. The method has been applied to the reaction of chymase and chymotrypsin with the tetrapeptide aldehyde, chymostatin, basic pancreatic trypsin inhibitor and Ala-Ala-Phe-chloromethylketone (AAPCK). The irreversible inhibitor, AAPCK, showed the expected saturation kinetics for both enzymes and the apparent first-order rate constants ( k 2) and dissociation constants ( K i) for the non-covalent complexes were determined. Chymostatin was a much more potent inhibitor which failed to show a saturation effect. The second-order rate constant of inactivation ( k 2/ K i), the first-order reactivation rate constant ( k −2), and the dissociation constant of the covalent complex ( K i ∗ ) were determined. Basic pancreatic trypsin inhibitor, a potent inhibitor of chymotrypsin, had similar kinetics to chymostatin but failed to inhibit chymase. The applicability of the two-step model and the integrated equation to slow- and tight-binding inhibitors is discussed in relation to a number of examples from the literature.

Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase.

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.

Identification of active-site residues of sheep liver serine hydroxymethyltransferase.

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

Inactivation of rat ovarian 20α-hydroxysteroid dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides, varying in chain length from N-ethylmaleimide and N-butyl to N-octyl, inclusive, was shown to effectively inactivate rat ovarian 20α-hydroxysteroid dehydrogenase at pH 7.7, 25 °C. The apparent second-order rate constants for inactivation were observed to increase with increasing chain length of the N-alkylmaleimide used. Positive chain length effects were also indicated by the K d values for N-alkylmaleimides calculated from double-reciprocal plots resulting from the saturation kinetics observed in the inactivation reactions. The maximum rate constant for inactivation at enzyme saturation was 0.3 min −1 for each maleimide studied. NADP-and coenzyme-competitive inhibitors such as 3-aminopyridine adenine dinucleotide phosphate and various adenosine derivatives protected the enzyme against maleimide inactivation, whereas no protection was observed with the steroid substrate, 20α-hydroxypregn-4-en-3-one. The pH profile for maleimide inactivation indicated the involvement of an enzyme functional group with a p K a near 8.0. Sulfhydryl modification was also indicated by fluorescein mercuric acetate inactivation and titration experiments. Inactivation of the enzyme by a lysine-modifying reagent exhibited a pH profile differing from that observed in the maleimide inactivation process. It is proposed that N-alkylmaleimides inactivate the enzyme through covalent modification of sulfhydryl groups located in a nonpolar region of the enzyme.

Irreversible inactivation of human erythrocyte pyruvate kinase by 2,3-butanedione

Human erythrocyte pyruvate kinase was found to be irreversibly inactivated by butanedione in the dark. The second-order rate constants for inactivation at pH 8.0 and 25 °C were 2.14 and 2.74 m −1 min −1 in the absence and presence of 50 m m borate, respectively. The pH profile of the inactivation indicated the involvement of a residue with an apparent p K a of 8.1–8.3. ADP and phosphoenolpyruvate acted as partial inhibitors of the inactivation process. Certain details of the inactivation, spectral studies, and fluorometric determinations gave evidence for arginine as the only target residue. A total of 23 ± 3 residues per subunit were modified within the period required for inactivation. In the same period the presence of 4 m m ADP reduced the extent of inactivation by 70% and the number of modified residues to 18 ± 4. The number of the arginine residues protected by ADP from butanedione modification was 5.0 ± 1.3 per subunit.

Essential histidyl residues in glucose oxidase. Chemical modification of histidyl residues with diethylpyrocarbonate.

The apoenzyme of glucose oxidase (EC 1.1.3.4) from Aspergillus niger was modified by diethylpyrocarbonate (DEPC). A second-order rate constant of inactivation with respect to the regaining of activity when reconstituted with FAD was 33.7M-1s-1 at pH 6.2 and 25°C. This inactivation was reversed up to 60% by the addition of 0.25 M hydroxylamine. It was suggested that histidyl residues having pKa = 8.2 were responsible for the reconstitution reaction of holo-oxidase from its pH-dependence of inactivation. In addition to these histidyl residues, two SH-groups were particitated in the DEPC modification, and these residues seemed likely to interact with each other. From the studies on the enzyme kinetics, CD spectra, and stoichiometry on the modification of histidyl residues, all data suggested that inactivation of apoenzyme with DEPC was due primarily to the modification of two histidyl residues essential for the binding of FAD and two cysteines near these residues. The present investigation suggested that there were some other histidyl residues necessary for maintaining the proper conformation of native glucose oxidase.

Neutral and acid proteinases from free living nematode Turbatrix aceti. Partial purification and characterization

1. 1. Neutral and acid proteinases from Turbatrix aceti were isolated and partially purified, 400–500-fold and characterized. 2. 2. Neutral proteinase is a thiol proteinase with a molecular weight of 22000 and pH optimum of 6.5. It exhibits chymotrypsin-like specificity and is inhibited by p-mercuribenzoate and β-mercaptoethanol. 3. 3. Neutral proteinase is also inhibited by a specific irreversible inhibitor of serine proteinases—diisopropylphosphofluoridate with second-order rate constant of inactivation = 21 min −1 M −1. However, it is not effected by high molecular weight inhibitors of serine proteinases. 4. 4. Acid proteinase is a typical carboxyl proteinase with a molecular weight of 60,000 and pH optimum of 3.5. It is inhibited by a specific inhibitor of carboxyl proteinases 1,2-epoxy-3-( p-nitrophenoxy) propane.

A study of the ionic properties of the essential histidine residue of yeast alcohol dehydrogenase in complexes of the enzyme with its coenzymes and substrates

1. Initial-rate studies of the reduction of acetaldehyde by NADH, catalysed by yeast alcohol dehydrogenase, were performed at pH 4.9 and 9.9, in various buffers, at 25 degrees C. The results are discussed in terms of the mechanism previously proposed for the pH range 5.9-8.9 [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. 2. Acetaldehyde forms a u.v.-absorbing complex with glycine. This was shown not to affect the results of kinetic experiments under the conditions used in this and earlier work. 3. The variation with pH of the dissociation constant for the enzyme-NADH complex, calculated from the initial-rate data, indicates that the enzyme possesses a group with pK7.1 in the free enzyme and pK8.7 in the complex. 4. The pH-dependences of the second-order rate constants for inactivation of the enzyme by diethyl pyrocarbonate were determined for the free enzymes (pK7.1), the enzyme-NAD+ complex (pK approx. 7.1) and the enzyme-NADH complex (pK approx. 8.4). The essential histidine residue may therefore be the group involved in formation and dissociation of the enzyme-NADH complex. 5. Estimates of the rate constant for reaction of acetaldehyde with the enzyme-NADH complex indicate that acetaldehyde may combine only when the essential histidine residue is protonated. The dissociation constants for butan-1-ol and propan-2-ol, calculated on the basis of earlier kinetic data, are, however, independent of pH. 6. The results obtained are discussed in relation to the role of the essential histidine residue in the mechanism of formation of binary and ternary complexes of the enzyme with its coenzymes and substrates.

Maleimideinactivation of lactate dehydrogenase isozymes

The homologous beef lactate dehydrogenase (EC 1.1.1.27) isozymes H 4 and M 4 were observed to be effectively inactivated by a homologous series of N- alkylmaleimides . With each isozyme, the second-order rate constants of inactivation increased with increasing chain length of the alkyl group of the maleimide derivative. The rate of inactivation of lactate dehydrogenase (H 4) by N- heptylmaleimide was decreased in the presence of NADH while no protective effect by NADH was noted in the inactivation of lactate dehydrogenase (M 4) by this maleimide. Adenosine, AMP, ADP and adenosine diphosphoribose were shown to be coenzyme-competitive inhibitors of both lactate dehydrogenase (H 4) and lactate dehydrogenase (M 4). Binding patterns of these compounds were very similar with the two isozymes studied.

On the irreversible inhibition of α-chymotrypsin, trypsin, pancreatic kallikrein', thrombin and elastase by N-( β-pyridylmethyl)-3,4 dichlorophenoxyacetamide- p-fluorosulfonylacetanilide bromide

1. 1. Kinetic parameters and specificity characteristics have been determined for N-( β-pyridylmethyl)-3,4-dichlorophenoxyacetamide- p-fluorosulfonylacetanilide bromide (PDFA), a member of a new class of active-site-directed irreversible protease inhibitors where covalent bonding to the enzymes occurs outside the limits of the active site proper. The results establish PDFA as one of the most potent inhibitors of α-chymotrypsin (EC 3.4.4.5) with a second-order rate constant of inactivation of 5.41 · 10 4 M −1 · min −1 at pH 7.0 and 37 °C. 2. 2. PDFA is also a weak irreversible inhibitor of trypsin (EC 3.4.4.4), pancreatic kallikrein (EC 3.4.4.21), elastase (EC 3.4.4.7) and thrombin (EC 3.4.4.13), in this approximate descending order of effectiveness. The second-order rate constant of inhibition with trypsin (at pH 8.1) is more than 200 times lower than with α-chymotrypsin (at pH 7.0).