Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde

Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.     

Inactivation of fructose-1,6-bisphosphatase by o-phthalaldehyde

Rabbit liver fructose-1,6-bisphosphatase, a tetramer of identical subunits was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The second-order rate constant for the inactivation was 30 M-1s-1. Fructose-1,6-bisphosphatase was completely protected from inactivation by the substrate--fructose-1,6-diphosphate but not by the allosteric effector--adenosine monophosphate. The absorption spectrum (lambda max 337 nm) and, fluorescence excitation (lambda max 360 nm) and fluorescence emission spectra (lambda max 405 nm) were consistent with the formation of an isoindole derivative in the subunit between a cysteine and a lysine residue about 3A apart. About 4 isoindole groups per mol of the bisphosphatase were formed following complete loss of the phosphatase activity. This suggests that the amino acid residues of the biphosphatase participating in reaction with o-phthalaldehyde more likely reside at or near the active site instead of allosteric site. The molar transition energy of fructose-1,6-bisphosphatase--o-phthalaldehyde adduct was estimated 121 kJ/mol and compares favorably with 127 kJ/mol for the synthetic isoindole, 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl) isoindole in hexane. It is, thus, concluded that the cysteine and lysine residues participating in isoindole formation in reaction between fructose-1,6-bisphosphatase and o-phthalaldehyde are located in a hydrophobic environment.     

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.     

Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a ''half-of-the-sites'' mechanism.

Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nature of o-phthalaldehyde reaction with pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase (FAS) was inactivated irreversibly by stoichiometric concentration of o-phthalaldehyde exhibiting a bimolecular kinetic process. FAS-o-phthalaldehyde adduct gave a characteristic absorption maxima at 337 nm. Moreover this derivative showed fluorescence emission maxima at 412 nm when excited at 337 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The inactivation is caused by the reaction of the phosphopantetheine -SH group since it is protected by either acetyl- or malonyl-CoA. The enzyme incubated with iodoacetamide followed by o-phthalaldehyde showed no change in fluorescence intensity but decrease in intensity was found in the treatment of 2,4,6-trinitrobenzenesulphonic acid (TNBS), a lysine specific reagent with the enzyme prior to o-phthalaldehyde addition. As o-phthalaldehyde did not inhibit enoyl-CoA reductase activity, so nonessential lysine is involved in the o-phthalaldehyde reaction. Double inhibition experiments showed that 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a thiol specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 moles of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation.     

Sequential inactivation of zeta-crystallin by o-phthalaldehyde

o-Phthalaldehyde, a bifunctional cross-linking reagent, is commonly used as a probe for the active site of enzymes. In this study, the interaction of o-phthalaldehyde with camel lens zeta-crystallin was examined by activity and fluorescence measurements. Predictably, the oxidoreductase activity of zeta-crystallin was inhibited irreversibly by o-phthalaldehyde in a time- and concentration-dependent manner, and the presence of NADPH with the enzyme appeared to provide a high degree of protection against o-phthalaldehyde inactivation. Interaction of o-phthalaldehyde with zeta-crystallin resulted in formation of isoindole adduct, which exhibited characteristic fluorescence at 415 nm. However, neither inactivation nor modification of the enzyme showed the expected pseudo-first-order kinetics; both events were highly sequential reaching different levels of saturation at different concentrations of o-phthalaldehyde. The modified enzyme had a maximum stoichiometry of 1 mol isoindole/subunit, and bound NADPH to nearly the same extent as unmodified enzyme. Gel filtration experiments suggested that o-phthalaldehyde-modified zeta-crystallin had higher apparent molecular weight than unmodified enzyme, even though the enzyme remained largely monomeric as revealed by electrophoresis on denaturing gel. These results suggested that modification by o-phthalaldehyde might have been so intrusive as to sequentially modify the tetrameric structure of zeta-crystallin.     

Effect of sulfhydryl group modification on the activity of bovine ferrochelatase

The role of sulfhydryl groups in the activity of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), has been examined by using a variety of sulfhydryl group-specific reagents. The enzyme is rapidly inactivated in a pseudo-first order reaction by N-ethylmaleimide and monobromobimane and more slowly by iodoacetamide and bromotrimethylammoniobimane. Reaction with [3H]N-ethylmaleimide indicates that modification of a single sulfhydryl group is sufficient to inactivate bovine ferrochelatase. The enzyme is protected from inactivation by one substrate, ferrous iron, but not by the porphyrin substrate. Mercury and arsenite are reversible inhibitors. The fluorescence of the bound bimane is blue shifted 8 nm from that obtained in aqueous solutions and is sensitive to quenching by iodide.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by o-phthalaldehyde.

The two activities of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by o-phthalaldehyde. Absorbance and fluorescence spectra of the modified enzyme were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme subunit). The inactivation of 6-phosphofructo-2-kinase by o-phthalaldehyde was faster than the inactivation of fructose-2,6-bisphosphatase, which was concomitant with the increase in fluorescence. The substrates of 6-phosphofructo-2-kinase did not protect the kinase against inactivation, whereas fructose-2,6-bisphosphate fully protected against o-phthalaldehyde-induced inactivation of the bisphosphatase. Addition of dithiothreitol prevented both the increase in fluorescence and the inactivation of fructose-2,6-bisphosphatase, but not that of 6-phosphofructo-2-kinase. It is proposed that o-phthalaldehyde forms two different inhibitory adducts: a non-fluorescent adduct in the kinase domain and a fluorescent isoindole derivative in the bisphosphatase domain. A lysine and a cysteine residue could be involved in fructose-2,6-bisphosphate binding in the bisphosphatase domain of the protein.     

Chemical modification of bovine heart mitochondrial malate dehydrogenase. Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.     

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.     

Affinity labeling of a subsite of Taka-amylase A by the fluorescent reagent o-phthalaldehyde

A cross-linked modification of Lys residue located at the subsite of the enzyme active site of Taka-amylase A was attained by the use of the fluorescent reagent of o-phthalaldehyde (OPA). The fluorescence and uv absorption at 337 nm derived from the isoindole ring, which was produced by cross-linking through the epsilon-amino group of Lys and the thiol group of the Cys residue, provided the evidence for the OPA-mediated inactivation of Taka-amylase A. Kinetic analysis showed that 1 mol of OPA per mole of enzyme was incorporated, which corresponded closely with the value obtained by the uv absorption. Because the OPA inactivation was retarded by the substrate analog of alpha-cyclodextrin, OPA modification was classified as a type of affinity labeling reaction. A remarkable increase in the pI value from 4.0 to 5.6 upon the modification led to clear separation of the modified enzyme from the native Taka-amylase A by a DEAE-Sephacel column and led to the charge isomer pattern on gel electrophoresis performed according to the method of Hedrick and Smith. Moreover, the affinity gel electrophoresis showed that the modified enzyme completely lost the affinity for the substrate soluble starch, which indicated that the subsite modification occurred.     

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.     

Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde

Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins.     

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.     

Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents. Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation. The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), with a binding stoichiometry of approx. 2 mol per mol of enzyme subunit. A high level of pyrene excimer fluorescence is detected on the labeled enzyme, thus implying the reaction of the reagent with two spatially close sulfhydryl groups in the protein. The carboxykinase is not completely inactivated by different vicinal dithiol-specific reagents, thus implying a catalytically non-essential character for these groups. From substrate protection experiments of the enzyme inactivation by DTNB, PyrIAM and vicinal dithiol-specific reagents, it is concluded that the loss of enzyme activity is caused by the modification of both thiol and vicinal dithiol groups in the substrate binding region.     

Conformation and polarity of the active site of xylanase I from Thermomonospora sp. as deduced by fluorescent chemoaffinity labeling. Site and significance of a histidine residue.

A fluorescent chemoaffinity label o-phthalaldehyde (OPTA) was used to ascertain the conformational flexibility and polarity at the active site of xylanase I (Xyl I). The kinetics of inactivation of Xyl I with OPTA revealed that complete inactivation occurred due to the binding of one molecule of OPTA to the active site of Xyl I. The formation of a single fluorescent isoindole derivative corroborated these findings. OPTA has been known to form a fluorescent isoindole derivative by crosslinking the proximal thiol and amino groups of cysteine and lysine. The involvement of cysteine in the formation of a Xyl I-isoindole derivative has been negated by fluorometric and chemical modification studies on Xyl I with group-specific reagents and by amino-acid analysis. The kinetic analysis of diethylpyrocarbonate-modified Xyl I established the presence of an essential histidine at or near the catalytic site of Xyl I. Modification of histidine and lysine residues by diethylpyrocarbonate and 2,4,6-trinitrobenzenesulfonic acid, respectively, abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating that histidine and lysine participate in the formation of the isoindole complex. A mechanism for the reaction of OPTA with histidine and lysine residues present in the protein structure has been proposed. Experimental evidence presented here suggests for the first time that the active site of Xyl I is conformationally more flexible and more easily perturbed in the presence of denaturants than the molecule as a whole. The changes in the fluorescence emission maxima of a model compound (isoindole adduct) in solvents of different polarity were compared with the fluorescence behaviour of the Xyl I-isoindole derivative, leading to the conclusion that the active site is located in a microenvironment of low polarity.     

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.     

Pig kidney dopa decarboxylase: inactivation by iodoacetamide and sequence of the carboxyamidomethylcysteine-containing peptide

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by iodoacetamide following pseudo-first order reaction kinetics. The apparent first order rate constant for inactivation is proportional to the concentration of iodoacetamide and a second order rate constant of 37 M-1 min-1 is obtained at pH 6.8 and 25 degrees C. Cyanogen bromide fragmentation of iodo(1-14C)acetamide - modified inactivated Dopa decarboxylase followed by trypsin digestion yields a single radioactive peptide. Automated Edman degradation reveals a heptapeptide sequence which contains labeled carboxyamidomethylcysteine. This finding and the results of the incorporation of the label from ido (1-14C)acetamide into the enzyme clearly indicate that the modification of 1 mol of SH per mol of enzyme dimer is responsible for the inactivation process. The labeled peptide, which was located by means of limited proteolysis on the fragment corresponding to the COOH-terminal third of the enzyme, has been aligned with a 7 amino acid stretch of Drosophila enzyme. Although this region appears highly conserved in the Dopa decarboxylase enzymes, the cysteinyl residue is not conserved. This observation together with the spectral binding properties of the iodoacetamide inactivated enzyme argue against a functional role for the modifiable cysteine in the mechanism of action of pig kidney enzyme. It is suggested that the loss of pig kidney decarboxylase activity produced by iodoacetamide modification might be attributable to steric hindrance. This could be due to the presence of the bulky acetamidic group on a cysteine residue at, or near, the active center or in a site of strategic importance to the maintenance of the active site topography.     

Reaction of rat liver glycine methyltransferase with 5'-p-fluorosulfonylbenzoyladenosine

Rat liver glycine methyltransferase is inactivated by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) in a pseudo-first order fashion at pH 7.5. The addition of dithiothreitol (20 mM) to the reaction mixture results in partial restoration of enzyme activity. A semilog plot of residual activity after dithiothreitol reactivation versus time is also linear, indicating that at least two essential residues are present on the enzyme and the modification of either of which causes total loss of activity. The inactivation is accompanied by incorporation of the radiolabel from adenine-labeled FSBA, but the amount of radioactivity fixed is not altered upon treatment with dithiothreitol. From this fact and the stoichiometry between the loss of dithiothreitol-sensitive activity and the number of sulfhydryl groups disappeared, it is suggested that the dithiothreitol-sensitive inactivation is the consequence of the FSBA-mediated formation of a disulfide between two sulfhydryl groups in close proximity. Although 4 mol of reagent are covalently bound per enzyme subunit, the kinetics of modification and inactivation show that the reaction at 1 residue, which is identified as tyrosine, is responsible for the dithiothreitol-insensitive inactivation. The substrate S-adenosylmethionine provides complete protection against both types of inactivation, but the dithiothreitol-insensitive inactivation is protected much more effectively with a Kd value comparable to the Km value. This suggests that the tyrosine is located at or near the active site of the methyltransferase.