Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.     

Pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a. Covalent modifications of aspartic acid 191, lysine 155, and the pyruvoyl group

The pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a is rapidly inactivated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and glycine ethyl ester. On 90% of inactivation, 1.3 residues of [14C]glycine ethyl ester are incorporated per alpha subunit; nearly 60% of this is linked to the beta-carboxyl group of Asp-191. Histamine, a competitive inhibitor, protects against this inactivation. The KM value of the modified enzyme for histidine (6.2 mM) is much higher than that of the unmodified enzyme (KM = 0.4 mM); catalytic activity is reduced but not eliminated. Thus, Asp-191 is the most reactive accessible carboxyl group under these conditions and is close to the substrate-binding site, but apparently is not essential for catalysis. At pH 8.0, fluorodinitrobenzene inactivates histidine decarboxylase completely with the incorporation of two dinitrophenyl residues/alpha subunit; the modified residues are Lys-155 and Cys-228. Urocanic acid, a competitive inhibitor, protects against inactivation. Treatment with mercaptoethanol restores the free -SH of Cys-228 but does not restore activity. Conversion of Cys-228 to its cyano derivative slows but does not prevent dinitrophenylation of Lys-155; the resulting derivative is catalytically inactive. Thus, Lys-155 is located within the active site and may play an essential role in catalysis. Finally, histidine methyl ester was shown to inhibit this decarboxylase by forming a Schiff's base with the essential pyruvoyl group.     

Chemical modification of Corynebacterium sarcosine oxidase: role of sulfhydryl and histidyl groups

Sarcosine oxidase [sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1] from Corynebacterium contained 8 sulfhydryl groups per mol of enzyme as determined with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 0.2% SDS and by titration with p-chloromercuribenzoate (PMB). Among them, 2 groups were easily modified by iodoacetamide (IAA) and the modification resulted in complete loss of enzymatic activity. The inactivation by IAA followed first-order kinetics with respect to IAA concentration. The presence of acetate, a competitive inhibitor (I), protected the enzyme from inactivation by IAA. However, the protection was only approximately 50%. The enzyme was also inactivated by PMB, but in this case, there was practically no recovery of activity after treatment with thiol compounds. The enzyme was also rapidly inactivated by incubation with diethylpyrocarbonate (DEP). The absorbance change accompanying the inactivation showed that a single histidyl residue was modified by DEP, resulting in a complete loss of enzymatic activity. In the presence of acetate, the enzyme was completely protected from DEP-inactivation. Furthermore, DEP-inactivated enzyme recovered its enzymatic activity on treatment with hydroxylamine. These observations seem to imply that the modified histidine is essential for enzyme activity. In addition, modification by DEP changed the absorption spectrum in the visible region. This strongly suggests that the modified histidyl residue is present in the vicinity of the flavin moiety of the enzyme molecule.     

Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.     

Selective carboxymethylation of cysteine-174 of the beta 2 beta 2 and beta 1 beta 1 human liver alcohol dehydrogenase isoenzymes by iodoacetate

The beta 1 beta 1 and beta 2 beta 2 human liver alcohol dehydrogenase isoenzymes differ by only one residue at the coenzyme-binding site; Arg-47 in beta 1 is replaced by His in the beta 2 subunit. Since Arg-47 is thought to facilitate the carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase by binding halo acids in a Michaelis-Menten complex prior to inactivation, the specificity and kinetics of modification of the two human liver beta beta isoenzymes with iodoacetate were compared. Both of the beta beta isoenzymes were inactivated by treatment with iodo[14C]acetate, and one Cys per subunit was carboxymethylated. Cys-174, which is a ligand to the active-site zinc atom in horse liver alcohol dehydrogenase, was selectively carboxymethylated in each of the human beta beta isoenzymes; less than 15% of the iodo[14C]acetate incorporated into the enzyme appeared in Cys-46. Therefore, the three-dimensional structure of the basic amino acids in the anion-binding site of the human beta beta isoenzymes appears to be different from that of horse liver alcohol dehydrogenase. The kinetics of alkylation are consistent with the formation of a Michaelis-Menten complex before inactivation of the isoenzymes. The average Ki values for iodoacetate were 10 and 16 mM for beta 1 beta 1 and beta 2 beta 2, respectively, and maximal rate constants for inactivation were 0.22 and 0.17 min-1, respectively. From these data, it can be concluded that there is a relatively minor effect of the substitution of His for Arg at position 47 on the kinetics of inactivation.     

The guanosine 3',5'-bis(diphosphate) (ppGpp) cycle. Comparison of synthesis and degradation of guanosine 3',5'-bis(diphosphate) in various bacterial systems.

4-(3-Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl-14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme-coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C-labelled coenzyme analogue and subseqeuntly carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys-38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys-43 in yeast alcohol dehydrogenase and Cys-46 in the horse liver enzyme but, unlike the latter two, Cys-38 is not reactive towards iodoacetate in the native bacterial enzyme.     

5,5′-Dithiobis-(2-Nitrobenzoic Acid) as a Probe for a Non-Essential Cysteine Residue at the Medium Chain Acyl-Coenzyme a Dehydrogenase Binding Site of the Human ‘Electron Transferring Flavoprotein’ (ETF)

Abstract

Human ‘electron transferring flavoprotein’ (ETF) was inactivated by the thiol-specific reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase.

1. The type III variant of chloramphenicol acetyltransferase (CATIII) is resistant to inactivation by ionizable modifying reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and iodoacetate, whereas it is sensitive to inhibition by similar but uncharged reagents, including 4,4'-dithiodipyridine, methyl methanethiolsulphonate (MMTS) and iodoacetamide. The target for these thiol-modifying reagents has been postulated to be Cys-31. This residue is situated within a part of the chloramphenicol-binding site formed largely from the side chains of hydrophobic amino acid residues, which might be expected to discriminate against the access of ionized ligands to Cys-31. 2. The substitution of Cys-31 by alanine, serine, threonine or methionine yields an enzyme that is resistant to inactivation by thiol-specific reagents. Replacement of Cys-31 by alanine, serine or threonine results in increased Km values for chloramphenicol with only small changes in kcat.. In contrast, the Cys-31----Met substitution mainly affects kcat. values. Although the kcat. for chloramphenicol acetylation is decreased 13-fold compared with wild-type CAT, the kcat. for the acetyl-CoA hydrolysis reaction, which occurs in the absence of chloramphenicol, is increased 2.7-fold. 3. MMTS modification of cysteine residues results in an adduct (-CH2-S-S-CH3) that is structurally similar to the side chain of a methionine residue (-CH2-CH2-S-CH3). The kinetic properties of MMTS-modified CATIII closely resemble those of [Met31]CAT.     

Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis

Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents.     

Spinach leaf ribulose-5-phosphate kinase: examination of sulfhydryls by chemical modification and spin-labeling

Methodology has been developed for complete or selective modification of the cysteinyl sulfhydryls of ribulose-5-phosphate (Ru5P) kinase. Using native enzyme, iodoacetate modifies four sulfhydryls with varying levels of completeness. The most reactive sulfhydryl in the native enzyme can be selectively titrated with iodoacetate; complete loss of activity occurs. Composition and N-terminal analyses of the peptide bearing this essential sulfhydryl indicate that the alkylated residue (Cys-16) is identical to the site modified by other modification reagents (M. A. Porter and F. C. Hartman (1986) Biochemistry 25, 7314-7318). In the presence of ATP, a nonessential sulfhydryl of the native enzyme is carboxymethylated. The peptide bearing this modified cysteine has been isolated and its composition and N-terminal sequence determined. Enzyme that is carboxymethylated in the presence of ATP retains activity and can be oxidatively inactivated in a reversible fashion. This suggests that the cysteine targeted by iodoacetate in the presence of ATP is not a residue that participates in regulation of enzyme activity. Using a spin-labeled analog of iodoacetate, both essential and nonessential cysteines have been selectively modified. ESR measurements suggest that the environment of these cysteines is not highly constrained. Modest effects on spin-label mobility are observed upon occupancy of Ru5P or ATP sites on the modified enzyme. These effects are dependent on the presence of divalent cations, suggesting that a binary enzyme-cation complex must form prior to productive enzyme-substrate interactions.     

Number and function of sulphydryl groups of N-acetylneuraminate lyase

The reaction between DTNB and the SH groups of N-acetylneuraminate lyase has been investigated in the presence and absence of pyruvic acid, substrate of the enzyme. It was found that DTNB inactivates N-acetylneuraminate lyase, while pyruvic acid protects the enzyme against this inactivation. When the enzyme was fully inactivated, two SH groups have reacted with DTNB. This result supports previous suggestions, that there is one cystein residue per active site responsible for enzyme activity. In the presence of SDS, approx. 6 SH groups reacted with DTNB suggesting the existence of 3 SH groups per enzyme subunit.     

Catalytic nonessentiality of an active-site cysteinyl residue of phosphoribulokinase

The activity of the Calvin cycle enzyme phosphoribulokinase is coupled to photosynthetic electron transport by reversible oxidation/reduction mediated by thioredoxin-f. Previous studies have shown that one of the regulatory sulfhydryl groups, that of Cys-16, is positioned at the nucleotide-binding domain of the active site. To determine if oxidative deactivation of the kinase reflects catalytic essentiality of Cys-16, the methylation of spinach phosphoribulokinase by methyl-4-nitrobenzenesulfonate has been examined. Methylation of the kinase results in a 50% loss of the initial activity relative to controls. The suppression of kcat is accompanied by a 6-fold increase in the Km for ATP, without change in the Km for ribulose 5-phosphate. The insensitivity of the modified enzyme, in contrast to the native, to iodoacetate and 5,5'-dithiobis(2-nitrobenzoate) indicates that Cys-16 is a site of methylation. This supposition is verified independently by peptide mapping and Edman degradation subsequent to S-carboxymethylation with [14C]iodoacetate of the methylated kinase. Retention of significant enzymatic activity after complete modification of Cys-16 with the small, uncharged methyl moiety demonstrates that this active-site residue is not essential for catalysis.     

Localization of the 5-phospho-α-d-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase

Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-α-d-ridosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [¹⁴C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [¹⁴C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the K_m for PRib-PP, but no change in V_max. Storage of HPRT also resulted in an increase in the K_m for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in K_m for PRib-PP. The stoichiometry of the reaction of [¹⁴C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups 5,5′-dithiobis(2-nitro-benzoic acid)] and the effect of dithiothreitol on K_m for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1mM β-mercaptoethanol.     

Chemical modifications of ribonuclease U1.

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.     

Inactivation of DNase by 2-nitro-5-thiocyanobenzoic acid. II. Serine and threonine are the sites of reaction on the DNase molecule.

The amino acid compositions of various fragments isolated from DNase treated with 2-nitro-5-thiocyanobenzoic acid (NTCB) show peptide bond cleavages to be at Thr14, Ser40, and Ser135. Isolation and characterization of radioactive tryptic and chymotryptic peptides of [14C]cyano-DNase reveal four points of peptide bond cleavage; in addition to Thr14, Ser40, and Ser135, cleavage occurs at the amino end of Ser72. Approximately 2.8 mol of [14C]cyano group are incorporated in the completely inactivated enzyme, in which 0.6 residue of Thr14, 0.8 of Ser40, and approximately 0.3 each of Ser72 and Ser135 are modified. The inactivation by NTCB can also be obtained by reacting the enzyme with a mixture of 5,5'-dithiobis(2-nitrobenzoic acid), KCN, and iodoacetate which generates NTCB. The mixture facilitates the uses of K[14C]N, which is readily incorporated into the enzyme as the [14C]cyano derivative. The reaction of NTCB with serine or threonine resembles that with cysteine.     

Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.     

Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP)

Phosphoenolpyruvate carboxykinase from the cytosol of rat liver has 13 cysteines, at least one of which is known to be very reactive and essential for catalytic activity (Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 17, 5329-5338). In order to identify the essential cysteine, this enzyme was modified with the fluorescent sulfhydryl reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Incubation of phosphoenolpyruvate carboxykinase with a 10% molar excess of this maleimide at 0 degrees C results in the rapid and nearly complete loss of catalytic activity. Under these conditions, 1 mol of the maleimide is incorporated per mol inactivated enzyme. The substrate GDP provides almost complete protection against inactivation and modification, while phosphoenolpyruvate protects against the rate, but not the extent, of modification. The pH dependence of the rate of enzyme inactivation suggests that the modified residue has a pK alpha of approximately 7.0. Purification and sequencing of the labeled peptide identifies the hyperreactive essential cysteine as Cys-288. This cysteine lies between two putative phosphoryl-binding domains and within a hydrophobic sequence.