Active Site-directed Reagent Research Articles

PH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5

The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure–function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five α- and ε-substituted l-Lys- d-Ala- d-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6–12.3, and the k cat/ K m vs. pH profile for activity against Ac 2- l-Lys- d-Ala- d-Ala was bell-shaped, with p K as at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed dd-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A β-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.

Modification of Human Erythrocyte Pyruvate Kinase by an Active Site-directed Reagent: Bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.

Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: identification of an essential cysteine residue

The mechanism of the l- threo-3-methylaspartate ammonia-lyase (EC 4.3.1.2) reaction has been probed using deuterium and solvent isotope effects with three different substrates, (2 S,3 S)-3-methylaspartic acid, (2 S)-aspartic acid and (2 S,3 R)-3-methylaspartic acid. Each substrate appears to form a covalent adduct with the enzyme through the amination of a dehydroalanine (DehydAla-173) residue. The true substrates are N-protonated and at low pH, the alkylammonium groups are deprotonated internally in a closed solvent-excluded pocket after K + ion, an essential cofactor, has become bound to the enzyme. At high pH, the amino groups of the substrates are able to react with the dehydroalanine residue prior to K + ion binding. This property of the system gives rise to complex kinetics at pH 9.0 or greater and causes the formation of dead-end complexes which lack Mg 2+ ion, another essential cofactor. The enzyme–substrate adduct is subsequently deaminated in two elimination processes. Hydrazines act as alternative substrates in the reverse reaction direction in the presence of fumaric acid derivatives, but cause irreversible inhibition in their absence. Borohydride and cyanide are not inhibitors. N-Ethylmaleimide also irreversibly inactivates the enzyme and labels residue Cys-361. The inactivation process is enhanced in the presence of cofactor Mg 2+ ions and Cys-361 appears to serve as a base for the removal of the C-3 proton from the natural substrate, (2 S,3 S)-3-methylaspartic acid. The dehydroalanine residue appears to be protected in the resting form of the enzyme by generation of an internal thioether cross-link. The binding of the substrate and K + ion appear to cause a conformational change which requires hydroxide ion. This is linked to reversal of the thioether protection step and generation of the base for substrate deprotonation at C-3. The deamination reaction displays high reverse reaction commitments and independent evidence from primary deuterium isotope effect data indicates that a thiolate acts as the base for deprotonation at C-3. 1 Bis Tris propane, 1,3-bis [tris(hydroxymethyl)-methylamino]propane; HPLC, high performance liquid chromatography; NEM, N-ethylmaleimide; NMR, nuclear magnetic resonance; UV, ultra-violet; TLC, thin layer chromatography; Tris, (Tris[hydroxymethyl]aminomethane); GC–MS, gas chromatography–mass spectrometry; SIR-GC–MS, single ion recording-gas chromatography–mass spectrometry. 1

Activation of pollen tube callose synthase by detergents. Evidence for different mechanisms of action.

In pollen tubes of Nicotiana alata, a membrane-bound, Ca(2+)-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-beta-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited by the membrane-impermeant, active site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity by the permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3-16, and 1-alpha-lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin.

Purification and Characterization of the Flavoprotein Tryptophan 2-Monooxygenase Expressed at High Levels in Escherichia coli

Tryptophan 2-monooxygenase from Pseudomonas savastanoi is a flavoprotein which catalyzes the formation of indoleacetamide from tryptophan. This is the first step in a two-step pathway for the formation of indoleacetic acid during infection of plants and subsequent gall formation by this and other bacteria. The enzyme has been expressed in Escherichia coli at high levels, and a purification procedure has been developed which generates micromolar amounts of protein. The purified enzyme contains tightly bound indoleacetamide; a method involving dialysis against 20% methanol has been developed for removing the indoleacetamide without significant loss of enzyme activity. Amino acids with large hydrophobic side chains are the best substrates. N-substituted phenylalanines will also act as substrates. N-ethylmaleimide, methyl methanethiol-sulfonate, and diethylpyrocarbonate act as active site-directed reagents, consistent with a histidine and a cysteine at or near the enzyme active site. Vinylglycine partially inactivates the enzyme, while propargylglycine has no effect.

3,4-Dihydro-3-methyl-6-nitro-2 H-1,3-benzoxazin-2-one, a Reagent for Labeling p-Nitrophenyl Esterases with a Chromophoric Reporter Group - Synthesis and Reaction with Chymotrypsin

We report the synthesis of 3,4-dihydro-3-methyl-6-nitro-2 H-l,3-benzoxazin-2-one (DMNB), a close structural analogue of p-nitrophenyl dimethylcarbamate. DMNB is unstable in aqueous solution when exposed to light, but is stable in the dark. The compound reacts slowly with chymotrypsin as an analogue of the substrate, p-nitrophenyl acetate, and this results in the incorporation of a p-nitrophenol-containing "reporter group" within the enzyme′s active site. The p K a of this group is two pH units higher than that of free p-nitrophenol, a result consistent with a hydrophobic environment around the enzymic binding site. Denaturation of the enzyme returns the p K a to its unperturbed value. DMNB has the promise of being a useful active site-directed reagent for other enzymes with p-nitrophenyl esterase activity.

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine.

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.

Intramolecular crosslinking of γ-glutamyl transpeptidase

γ-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor ( M r 78,000), glycosylated high molecular weight forms ( M r 85,000–95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-aminohippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent M r values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.

Evidence for catalytic site cysteine and histidine by chemical modification of β-hydroxy-β-methylglutaryl-coenzyme a reductase

S-(4-Bromo-2,3-dioxobutyl)-coenzyme A inactivates both yeast and rat liver beta-hydroxy-beta-methylglutaryl-coenzyme A reductase. The inactivation is irreversible, complete in 15 s, and proportional to the concentration of the reagent. beta-Hydroxy-beta-methylglutaryl-CoA provides protection against inactivation, whereas NADPH does not. Inactivation is attributed to reaction with an essential cysteine at the beta-hydroxy-beta-methylglutaryl-CoA binding site. Experiments with other active site-directed reagents confirm the involvement of a cysteine and support the presence of an active-site histidine, but rule out the participation of arginine or serine.

S-adenosylhomocysteinase from rat liver. Amino acid sequences of the peptides containing active site cysteine residues modified by treatment with 5'-p-fluorosulfonylbenzoyladenosine.

5'-p-Fluorosulfonylbenzoyladenosine (FSBA) inactivates rat liver S-adenosylhomocysteinase exhibiting characteristics of an active site-directed reagent. The inactivation is not associated with the covalent binding of the reagent, but is correlated with the loss of 2 sulfhydryl groups/enzyme subunit (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362). Treatment of the FSBA-inactivated enzyme with iodoacetate in the absence of reducing agent and then with [14C] iodoacetate after reduction with 2-mercaptoethanol yielded the enzyme containing approximately 2 mol of radiolabeled S-carboxymethylcysteine/mol of subunit. Analysis of tryptic peptides showed that the radioactivity was associated with 2 carboxymethylcysteine-containing peptides whose amino acid sequences were: Trp-Ser-Ser-Cys(Cm)-Asn-Ile-Phe-Ser-Thr-Gln-Asp-His-Ala-Ala-Ala-Ala-Ile- Ala-Lys and Gly-Glu-Thr-Asp-Glu-Glu-Tyr-Leu-Trp-Cys(Cm)-Ile-Glu-Gln-Thr-Leu-His-Phe- Lys, respectively. These results indicate that the inactivation of S-adenosylhomocysteinase by FSBA is the consequence of formation of a disulfide between two specific cysteine residues on each of the four identical subunits.

Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction.

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.

70] Modifiers of F1-ATPases and associated peptides

This chapter describes the modifiers of F1-ATPases and the associated peptides. The chemical modifiers of proteins can be divided into two classes—the group-directed reagents (chemical reagents capable of binding to side-chain residues in the investigated protein); and the active site-directed reagents (affinity and photoaffinity labels). The mitochondrial, bacterial, and chloroplast ATPases are H + -dependent ATPases (ATP synthases), as they catalyze both ATP hydrolysis and synthesis. Dicyclohexylcarbodiimide (DCCD) is used as a chemical reagent to block the proton conduction in the F 0 channel of the ATPase complex at the level of the DCCD-binding protein. Intermolecular cross-linking by random collisions between independent F 1 molecules is not predominant at the protein concentrations used for chemical modification. Intramolecular cross-linking can establish bridges in a single subunit (hairpin formation) or between two adjacent subunits but the cross-linking requires higher concentrations of reagents than inactivation.

Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO 2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A : Dependence on pH and effect of enzyme phosphorylation level

Steady-state kinetic parameters for the hydrolysis of the chromophoric hexapeptide Leu-Ser-Phe(NO 2)-Nle-Ala-Leu-OMe catalyzed by bovine gastricsin and pepsin A were determined. It was shown that the phosphate content of bovine pepsin A is without any significant effect on that parameters. At pH 4.7, the specificity constant ( k cat/ K m) was 2455 and 2150 mM −1 · s −1 for the most phosphorylated bovine pepsin A (2.58 phosphate groups per molecule), before and after treatment by potato acid phosphatase, respectively. The k cat/ K m ratio found for bovine gastricsin (1314 mM −1 · s −1) was closer to that of bovine pepsin A than that previously reported for chymosin (25 mM −1 · s −1). The spectral properties of the chromophoric tripeptide Leu-Ser-Phe(NO 2) in the pH range 1–3.6 were investigated. We have shown that the hexapeptide hydrolysis could be followed by difference spectrophometry at 295 nm ( Δϵ = −235 M −1 · cm −1 at pH 1.0) thus allowing to study the effect of pH on bovine pepsin A activity in a pH range which could not be explored earlier. The pH-dependence of k cat/ K m ratio of unphosphorylated bovine pepsin A indicated that enzyme activity was dependent upon the ionization of two groups of the enzyme whose p K are 1.2 and 5.0. These p K values strongly suggest the involvement of two carboxyl groups probably corresponding to the two reactive aspartyl residues (Asp 32 and Asp 215) identified through active site-directed reagents for all the aspartic proteinases so far tested.

The reaction mechanism of the mitochondrial pyridine nucleotide transhydrogenase. A study utilizing arylazido-pyridine nucleotide analogues.

The addition of a purified mitochondrial pyridine nucleotide transhydrogenase enzyme preparation to complex I (NADH-CoQ reductase) results in a significant increase in the NADPH-AcPyAD+ transhydrogenase activity of the complex without influencing the NADH-AcPyAD+ transhydrogenase activity. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of complex I, the purified transhydrogenase enzyme preparation was found to co-migrate with the Mr = 130,000 (130K) subunit of the NADH-CoQ reductase. Loss of the NADPH-NAD+ transhydrogenase activity of complex I following limited tryptic digestion was associated with a corresponding loss of the 130K subunit from the complex. These results suggest that the 130K subunit of complex I is the specific peptide responsible for the catalysis of the NADPH-NAD+ transhydrogenase activity observed in complex I. Studies have been carried out testing the influence of photoaffinity pyridine nucleotide probes on the NADPH-NAD+ transhydrogenase activity catalyzed at three levels of resolution, i.e. a homogeneous transhydrogenase preparation, a partially resolved membrane preparation (complex I), and an intact mitochondrial membrane preparation (EDTA particles). Such studies have revealed arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+) to be a potent inhibitor and an active site-directed reagent for NADPH-NAD+ transhydrogenation at all three levels of resolution. On the other hand, arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino]propionyl)NAD+ does not produce a significant degree of inhibition of NADPH-NAD+ transhydrogenase activities prior to or following photoirradiation. Nevertheless, the NAD+ analogue has been found to specifically label, covalently, the transhydrogenase protein following photoirradiation of an enzyme-analogue mixture. Arylazido-beta-alanyl NAD+ can as well function as a substrate during transhydrogenation by virtue of being able to accept a hydride ion from NADPH. An interpretation of the observed nucleotide photoprobe specificity for interaction at the active site for transhydrogenation is advanced. In this interpretation, an ordered binding of substrate involves an initial NADP(H) (or NADP+ photoprobe) interaction with a hydrophobic region at the transhydrogenation site. This initial reactivity is followed by a positioning of NAD(H) (or the NAD+ photoprobe analogue) above or periphery to the NADP(H) nucleotide present at the active site region. Supportive evidence for this model for transhydrogenation is presented and discussed.

Reaction of malate thiokinase with methoxycarbonyl-CoA disulfide. Evidence for half-of-the-sites reactivity.

Reaction of the active site-directed sulfhydryl reagent methoxycarbonyl-CoA disulfide with malate thiokinase follows pseudo-first order kinetics and leads to a complete loss in enzyme activity as measured by the overall reaction. The rate of inactivation of enzyme in buffer alone shows a linear dependence on the methoxycarbonyl-CoA disulfide concentration. In the presence of the substrate phosphate or the substrate analog sulfate, the rate of enzyme inactivation displays a hyperbolic response with respect to methoxycarbonyl-CoA disulfide. Anions such as chloride and fluoride decrease the rate of enzyme inactivation. Low concentrations of succinyl-CoA protect the enzyme against inactivation, while L-malate protection of the enzyme is observed only in the absence of added sulfate. Protection of the enzyme by ATP can be attributed to phosphorylation of the enzyme and a decrease in reactivity of the phosphoenzyme relative to the native enzyme. Enzyme reacted with excess methoxycarbonyl-CoA disulfide, although inactive with respect to turnover, catalyzes ATP-ADP exchange at one-half the rate of the native enzyme. The inactive enzyme can be phosphorylated by ATP, but only to 50% the extent of native enzyme. Titration of the enzyme with methoxycarbonyl-CoA disulfide shows that the enzyme exhibits all-of-the-sites reactivity with respect to this reagent. A correlation of titration of the enzyme with methoxycarbonyl-CoA disulfide with the loss in phosphorylation site shows that reaction of methoxycarbonyl-CoA disulfide produces half-of-the-sites reactivity with respect to enzyme phosphorylation.

Chemical modification of D-amino acid oxidase. Amino acid sequence of the tryptic peptides containing tyrosine and lysine residues modified by fluorodinitrobenzene.

D-Amino acid oxidase can be inactivated by covalent modification of predominantly tyrosine residue(s) at pH 7.4 by a low molar excess of fluorodinitrobenzene, which appears to act as an active site-directed reagent (Nishino, T., Massey, V., and Williams, C. H., Jr. (1980) J. Biol. Chem. 255, 3610-3616). Peptide mapping by high performance liquid chromatography of tryptic digests of protein modified with radiolabeled reagent revealed two major radioactive fractions with substantially different retention times which were not observed in protein modified in the presence of benzoate, a potent competitive inhibitor. Isolation and sequence analysis of the major radiolabeled peptides, as well as other direct chemical analyses, are presented which unambiguously demonstrate that these fractions represent modification of two different regions of the protein. The majority of the radiolabel was found within a 61-amino acid residue peptide containing an O-DNP-tyrosine residue exclusively at position 17. The substantial sequence surrounding this tyrosine residue indicates that it is different from that shown to react with N-chloro-D-leucine (Ronchi, S., Galliano, M., Minchiotti, L., Curti, B., Rudie, N. G., Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 6044-6046). The second fraction consisted of a 12-residue peptide containing an epsilon-DNP-lysine residue at position 5. Together, these two modified amino acids represented 0.89 mol of DNP incorporated/protein monomer. Both modifications must contribute to inactivation to account for the 90% decrease in enzymatic activity. Evidence is presented which suggests that both groups are within the active center of the enzyme and are modified in a mutually exclusive manner.

25] Application of affinity labeling for studying structure and function of enzymes

Publisher Summary This chapter discusses the uses of active-site directed reagents and considers the design and evaluation of such reagents for the benefit of those investigators who may wish to use this approach. In the characterization of an enzyme, it is interesting to try to identify amino acid residues that are at the active site and possibly involved in catalytic activity. An investigator may attempt to design and synthesize a new active-site-directed reagent. The active-sitedirected reagent can be used to determine how environmental factors affect reactivity. Thus the pH dependence of modification can be used to give pK values for the reactive amino acid, or at least for the system of which it is a part. The use of well-designed active-site-directed reagents that closely resemble a substrate, bind tightly to the enzyme, and react with considerable facilitation may also lead one to attempt to position the modifiable residue near a part of the substrate and to assign a catalytic role. In addition, they offer one of the best avenues for the rational development of specific chemotherapeutic agents.

Preparation and 13C NMR characterization of [[epsilon-13C] methionine-192]-alpha-chymotrypsin. The demethylation of [S-[13C] methylmethionine-192]-alpha-chymotrypsin by an active site-directed thiol.

Met-192 forms part of the binding crevice of alpha-chymotrypsin. The aim of this investigation was to find a nucleophile that would displace a methyl group from the sulfonium cation of [S-[13C]methylmethionine-192]-alpha-chymotrypsin without disrupting the five disulfide bridges of the protein, thereby producing [[epsilon-13C]methionine-192]-alpha-chymotrypsin, an isotopically enriched version of the native enzyme desirable for 13C NMR studies. Treatment of [S-methylmethionine-192]-alpha-chymotrypsin with mercaptoethanol and dithiothreitol failed to produce the latter protein, as deduced from elution profiles of reaction mixtures chromatographed on affinity columns of immobilized lima bean trypsin inhibitor. In contrast, when [S-methylmethionine-192]-alpha-chymotrypsin was incubated in a 3.0 mM solution of the active site-directed reagent 2-mercaptoacetyl-4'-methoxyanilide at pH 8.6 and 5 degrees C for 48 h, affinity chromatograms indicated the presence of a protein corresponding to native alpha-chymotrypsin. Upon repeating the experiment with [S-[13C]methylmethionine-192]-alpha chymotrypsin, we isolated in 40% yield a protein which was identified as [[epsilon-13C]methionine-192]-alpha-chymotrypsin by a combination of 13C NMR and chemical criteria. This work represents the first active site-directed demethylation of an S-[13C]methylmethionine residue at the binding site of an enzyme.

Effect of hydroxynitrobenzylation of tryptophan-177 on reactivity of active site cysteine-25 in papain

It is known that the enzymatic activity of papain (EC 3.4.22.2) toward α- N-benzoyl- l-arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4-nitrophenyl ( N-carbobenzoxy)glycinate ( S.-M. T. Chang and H. R. Horton, 1979, Biochemistry 18, 1559–1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactivation by S N2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloroacetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulfhydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the increased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, α- N-benzoyl- l-phenylalanyl- l-valyl- l-arginine p-nitroanilide, and toward an ester substrate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxynitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward α- N-benzoyl- l-arginine p-nitroanilide, apparently by blocking nonproductive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159).