Active site labeling of a receptor-like protein tyrosine phosphatase.

The inactivation of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase (PTPase), by iodoacetate and not by iodoacetamide suggested that iodoacetate interacts in a highly selective manner with the enzyme. The data indicate that iodoacetate binds at the active site of the enzyme with a stoichiometry of 0.8 mol of iodoacetate bound per mol of rat LAR. A single [14C]iodoacetate-labeled peptide was isolated following endoproteinase Lys-C digestion of the radiolabeled PTPase. Sequence analysis of the active site labeled peptide demonstrates that Cys-1522 contains the radiolabel. This residue has been shown by site-directed mutagenesis to be essential for rat LAR activity (Pot, D. A., Woodford, T. A., Remboutsika, E., Haun, R. S., and Dixon, J. E. (1991) J. Biol. Chem. 266, 19688-19696). Iodoacetate reacts only with the first domain of this double domain PTPase. These results, for the first time, directly identify the highly reactive cysteine residue at the active site of a PTPase and highlight the ability of this residue to participate as a nucleophile in the hydrolysis of phosphate from tyrosine.     

Identification of cysteine-319 as the target amino acid of 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate in bovine liver glutamate dehydrogenase.

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) has been shown to react with bovine liver glutamate dehydrogenase in the region of the GTP-dependent NADH inhibitory site with incorporation of about 1 mol of reagent/mol of subunit [Ozturk, D. H., Safer, D., & Colman, R. F. (1990) Biochemistry 29, 7112-7118]. The modified enzyme was shown to contain only 5 free sulfhydryl groups upon 5,5'-dithiobis (2-nitrobenzoate) titration as compared with 6 in the unmodified enzyme. In the unmodified enzyme digested with trypsin, 6 cysteinyl peptides were detected by high-performance liquid chromatography upon treatment with iodo [3H]acetic acid. In contrast, only 5 (carboxymethyl)cysteinyl peptides were detected in 8-BDB-TA-5'-TP-modified enzyme. When carboxymethylated modified and unmodified enzymes were digested with thermolysin, 6 peptide sequences containing (carboxymethyl)cysteine were obtained in the unmodified enzyme, but only 5 were observed in the modified enzyme. The (carboxymethyl)cysteine which was absent in the modified enzyme was determined to be Cys-319, leading to the conclusion that 8-BDB-TA-5'-TP reacts with Cys-319, thereby preventing it from subsequent reaction with radioactive iodoacetate. It was previously reported that 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TA-5'-DP) modifies Cys-319 in this enzyme [Batra, S. P., & Colman, R. F. (1986) Biochemistry 25, 3508-3515].(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase.

A single amino group in horse liver alcohol dehydrogenase was modified with methyl(14C)acetimidate by a differential labeling procedure. Lysine residues outside the active site were modified with ethyl acetimidate while a lysine residue in the active site was protected by the formation of an enzyme-NAD+-pyrazole complex. After the protecting reagents were removed, the enzyme was treated with methyl(14C)acetimidate. Enzyme activity was enhanced 13-fold as 1.1 (14C)acetimidyl group was incorporated per active site. A labeled peptide was isolated from a tryptic-chymotryptic digest of the modified enzyme in 35% overall yield. Amino acid composition and sequential Edman degradations identified the peptide as residues 219-229; lysine residue 228 was modified with the radioactive acetimidyl group.     

Reaction of p-azidophenacyl iodoacetate, a photolabile reagent, with yeast alcohol dehydrogenase.

When yeast alcohol dehydrogenase (YADH) was incubated with one or two molar equivalents of the photolabile reagent p-azidophenacyl iodoacetate (1), 10–15% of the enzymatic activity was lost per mole of inhibitor incorporated, a result which suggests 1 may be modifying in a cooperative process both the Cys-43 and the Cys-153 groups found at each active site of the enzyme. YADH incorporated a maximum of 5.6 mol of 1 per mole of enzyme. When YADH was first carboxymethylated and then allowed to react with an excess of 1, 3.2–3.6 mol of 1 were incorporated into the enzyme with a corresponding loss of 4.0 mol of free sulfhydryl groups in the enzyme. Carboxymethylated YADH was reacted with one molar equivalent of ¹⁴C-1 and then was treated sequentially with hydroxylamine and pepsin. Cellulose phosphate chromatography of this peptic digest gave one major radioactive peak eluting in the region where peptic peptides of YADH known to be modified at the Cys-153 are found to elute. When carboxymethylated YADH was treated with one molar equivalent of 1 and then photolyzed, at least 18% of the 1 residues became covalently bound to a second site in the enzyme. This finding establishes that 1 is a useful reagent for investigating the three-dimensional structure of the active site of YADH. Furthermore, 1 should be suitable for investigations into a variety of biological systems.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning, sequencing, overproduction, isolation, and characterization

The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi.     

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.     

Carboxymethylation of horse-liver alcohol dehydrogenase in the crystalline state. The active-site zinc region and general anion-binding site of the enzyme correlated in primary and teritiary structures.

Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)2-/4 , 1,10-phenanthroline, Au(CN)-/2 and AMP also prevented alkylation with decreasing effectiveness. Crystallographic studies of the alkylated enzyme show that the carboyxmethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN2-/4 to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom. The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN2-/4 and to a lesser extent Au(CN)-/2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc. Identificationof this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.     

Alkylation of cysteinyl residues of pig heart NAD-specific isocitrate dehydrogenase by iodoacetate.

Pig heart NAD-specific isocitrate dehydrogenase is inactivated by reaction with iodoacetate at pH 6.0. Loss of activity can be attributed to the formation of 1-2 mol of carboxymethyl-cysteine per peptide chain. The rate of inactivation is markedly decreased by the combined addition of Mn2+ and isocitrate, but not by alpha-ketoglutarate, the coenzyme NAD or the allosteric activator ADP. The substrate concentration dependence of the decreased rate of inactivation yields a dissociation constant of 1.6 mM for the enzyme-manganous-dibasic isocitrate complex, a value that is 50 times higher than the Km for this substrate. This result suggests that in protecting the enzyme against iodoacetate, isocitrate may bind to a region distinct from the catalytic site. Isocitrate and Mn2+ also prevent thermal denaturation, with an affinity for the enzyme close to that observed for the iodoacetate-sensitive site. The alkylatable cysteine residues may contribute to a manganous-isocitrate binding site which is responsible for stabilizing an active conformation of the enzyme.     

Binding of ligands to the catalytic zinc ion in horse liver alcohol dehydrogenase

The affinity of nitrogen and sulfur ligands for the catalytic zinc ion in horse liver alcohol dehydrogenase has been investigated by their influence on the affinity labeling reaction with iodoacetate. All the nitrogen compounds including ammonia, a primary and a secondary amine, and heterocycles containing a pyridine-type nitrogen with the exception of 2,2-dipyridyl were found to activate the affinity labeling reaction. Activation results from inner-sphere ligand coordination to the catalytic zinc ion. Closely related pyridine compounds gave a regular increase in affinity for the enzyme with increasing basicity, as expected for coordination to a metal ion. The sulfur compounds penicillamine and mercaptoethanol also activated the affinity labeling reaction, but dimercaptopropanol bound very tightly as a bidentate inhibited the reaction. The anions hydrosulfide, diethyldithiocarbamate, and cyanide coordinated to the catalytic zinc ion, whereas azide, thiocyanate, tetrazole, and iodide complexed the anion-binding site. The anionic metal ligands increased the rate of inactivation of the enzyme with iodoacetamide by binding to the catalytic zinc ion, while the binding of iodoacetate to the anion-binding site was prevented.     

Active site labeling of the shikimate pathway enzyme, dehydroquinase. Evidence for a common substrate binding site within dehydroquinase and dehydroquinate synthase

Dehydroquinase, the third enzyme of the shikimate biosynthetic pathway, is inactivated by iodoacetate. Iodoacetate behaves as an affinity label for the Escherichia coli enzyme with a Ki of 30 mM and a limiting inactivation rate of 0.014 min-1 at pH 7.0 and 25 degrees C. Affinity labeling is mediated by the negative charge of the reagent since iodoacetamide does not inactivate the enzyme. 2.1-2.3 mol of carboxymethyl groups are incorporated per mol of protein monomer resulting in 90% inactivation of enzymic activity. The majority of the bound label (80%) is split equally between 2 methionine residues, Met-23 and Met-205, which were identified by sequencing radiolabelled peptide fragments isolated after proteolytic digestion. An equilibrium mixture of the substrate (dehydroquinate) and product (dehydroshikimate) substantially reduces the inactivation rate and specifically decreases the incorporation of label at both of these site, implicating them as being in or near the active site of the enzyme. Sequence alignments with other biosynthetic dehydroquinases show that of the 2 methionine residues only Met-205 is conserved. N-terminal alignments of all the available dehydroquinase sequences (both catabolic and biosynthetic classes) revealed that Met-23, although itself not conserved, resides within a cluster of conserved sequence which may constitute part of the dehydroquinate binding site. A consensus sequence was derived from these alignments and used to probe the protein sequence data banks. A related sequence was found in dehydroquinate synthase, the enzyme which precedes dehydroquinase in the shikimate pathway. These results suggest that we have identified part of the dehydroquinate binding site in both enzymes.     

Cyanate modification of essential lysyl residues of the diphosphopyridine nucleotide-specific isocitrate dehydrogenase of pig heart.

The DPN-specific isocitrate dehydrogenase of pig heart is totally and irreversibly inactivated by 0.05 M potassium cyanate at pH 7.4 A plot of the rate constant versus cyanate concentration is not linear, but rather exhibits saturation kinetics, implying that cyanate may bind to the enzyme to give an enzyme-cyanate complex (K equal 0.125 M) prior to the covalent reaction. In the presence of manganous ion the addition of isocitrate protects the enzyme against cyanate inactivation, indicating that chemical modification occurs in the active site region of the enzyme. The dependence of the decrease of the rate constant for inactivation on the isocitrate concentration yields a dissociation constant for the enzyme-manganese-isocitrate complex which agrees with the Michaelis constant. The allosteric activator ADP, which lowers the Michaelis constant for isocitrate, does not itself significantly affect the cyanate reaction; however, it strikingly enhances the protection by isocitrate. The addition of the chelator EDTA essentially prevents protection by isocitrate and manganous ion, demonstrating the importance of the metal ion in this process. The substrate alpha-ketoglutarate and the coenzymes DPN and DPNH do not significantly affect the rate of modification of the enzymes by cyanate. Incubation of isocitrate dehydrogenase with 14C-labeled potassium cyanate leads to the incorporation of approximately 1 mol of radioactive cyanate per peptide chain concomitant with inactivation. Analysis of acid hydrolysates of the radioactive enzyme reveals that lysyl residues are the sole amino acids modified. These results suggest that cyanate, or isocyanic acid, may bind to the active site of this enzyme as an analogue of carbon dioxide and carbamylate a lysyl residue at the active site.     

The reaction of bovine thrombin with N-butyrylimidazole. Two different reactions resulting in the inhibition of catalytic activity.

N-Butyrylimidazole has been found to be a potent inhibitor of purified bovine thrombin. The rate and extent of inhibition of thrombin by N-butyrylimidazole could be reduced by the presence of benzamidine, a competitive inhibitor, or by the ester substrate, p-tosyl-L-arginine methyl ester. Spectral studies of the reaction of N-butyrylimidazole with thrombin demonstrated the modification of approximately 1 mol of tyrosine/mol of enzyme at maximum inhibition. In addition to the reaction with tyrosine, N-butyrylimidazole also appears to react with a residue at the "active site" as judged by a decrease in the number of active sites available in the modified enzyme for titration with p-nitrophenyl-p'-guanidinobenzoate. The time course of ester hydrolysis by butyrylated thrombin showed a distinct lag phase suggesting partial reactivation of the enzyme under assay conditions. Partial reactivation of the modified enzyme also occurred spontaneously upon standing in 0.5 M NaCl but was much faster in presence of imidazole (0.03 M, pH 7.6). It is suggested that, in addition to reaction with tyrosine, there is a reaction of N-butyrylimidazole with either the histidine and/or serine residue at the active site of thrombin resulting in a derivative unstable under esterase assay conditions such as that described for the reaciton of N-acetylimidazole with trypsin (L. L. Houston and K. A. Walsh (1970), Biochemistry 9, 156).     

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.     

Identification of the 5-iodoacetamidofluorescein reporter site on the Na,K-ATPase

5-Iodoacetamidofluorescein (5-IAF) labels the catalytic (alpha) subunit of dog kidney Na,K-ATPase without inhibiting enzymatic activity and is thus a useful fluorescent reporter of enzyme conformation under conditions of enzyme turnover. In this study conditions for labeling a unique sulfhydryl group are described, and this residue is identified in the cDNA-derived sequence. Reaction with iodoacetate (IAA) prior to fluorescent labeling lowers the stoichiometry of 5-IAF incorporation from 2.1 to 1.2 mol/mol alpha beta protomer, and increases the conformationally dependent fluorescence changes by 40-50%, consistent with the elimination of nonspecific labeling. IAA/IAF-enzyme has the same catalytic activity as the IAF-enzyme. In contrast, treatment with iodoacetamide prior to labeling with 5-IAF abolishes all fluorescence responses, although activity is retained. IAA/IAF-enzyme was digested by extensive trypsinolysis, and the fluorescent peptides released from the membrane were purified by high performance liquid chromatography and sequenced. Several fluorescent peptides were found, containing all or part of the sequence Cys-Ile-Glu-Leu-Cys-Cys-Gly-Ser-Val-Lys, corresponding to residues 452-461 in the sheep alpha subunit. The major site of modification is the second of the vicinal cysteine residues, Cys-457. Phenylarsine oxide, a reagent specific for vicinal sulfhydryl groups, prevents fluorophore incorporation, thereby confirming the identification of the IAF site from the sequence data.     

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.     

Amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins

The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val-Val-Gly-Gly-Trp-Asp-Phe-Val-Asp-Asn-Asp-Ser-Thr- Pro-Gln-Asn-Gly-Asn-Gly-64His-Gly-Thr-His-68Cys-Ala- Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn-Asn-Ser-Thr-Gly-Ile- Ala-Gly-Thr-Ala-Pro-Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-14CH2-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

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.     

Amino acid sequence at the active site of beta-glucosidase A from bitter almonds.

beta-Glucosidase A from bitter almonds was inhibited by the substrate analogue 6-bromo-3,4,5-trihydroxycyclo[2-3H]hex-1-ene oxide. Incorporation of 2 mol inhibitor/mol of dimeric enzyme resulted in total loss of activity. From tryptic digests of the labeled enzyme two radioactive peptides were isolated and their sequence determined (binding site of inhibitor underlined): peptide I, containing approx. 60% of the label: Ile-Thr-Glx-Glx-Gly-Val--Phe-Gly-Asp-Ser-Glx-(Ala, Asx2, Pro)-Lys and peptide II with approx. 30% of the label: Gly-Thr-Glx-Asp. The specifity of the reaction of beta-glucosidases (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with substrate-related epoxides indicates that the aspartic acid labeled in peptide I participates in the catalytic process of beta-glucoside hydrolysis. The labeling of a second site is interpreted in terms of two, mutually exclusive, binding modes of the inhibitor.