Processive action of the two peptide binding sites of prolyl 4-hydroxylase in the hydroxylation of procollagen

The number of peptide binding sites of prolyl 4-hydroxylase was manipulated with the peptide photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5, and the effect on hydroxylation of the relatively short peptide substrate (Pro-Pro-Gly)5 and of the long natural substrate procollagen was studied. With (Pro-Pro-Gly)5 as a substrate, a linear relation was found between enzyme activity and the amount of covalently bound photoaffinity label, approximately 50% inactivation being reached at 1 mol of label/mol of enzyme. No difference in Km value for (Pro-Pro-Gly)5 was detected between unlabeled and partially labeled enzyme preparations. These results indicate that enzyme molecules with only one free active site hydroxylated the synthetic substrate (Pro-Pro-Gly)5 with the same Km and at half the rate of native enzyme. In contrast, with procollagen as a substrate a 5-10-fold increase in Km was found with the fraction of enzyme containing only one free active site, as compared to the Km for procollagen with nonlabeled enzyme. This finding is explained by an enzyme-kinetic model based on a processive action of the two peptide substrate binding sites of prolyl 4-hydroxylase, preventing dissociation of the enzyme-substrate complex between successive hydroxylations of a long peptide with multiple substrate sites. Such a mechanism leads to a low Km for a long peptide by overcoming the diffusional constraints on the rate of association between the enzyme and the individual substrate sites.     

Identification of tyrosine 79 in the tocopherol binding site of glutathione S-transferase pi

Alpha-tocopherol, the most abundant form of vitamin E present in humans, is a noncompetitive inhibitor of glutathione S-transferase pi (GST pi), but its binding site had not been located. Tocopherol iodoacetate (TIA), a reactive analogue, produces a time-dependent inactivation of GST pi to a limit of 25% residual activity. The rate constant for inactivation, k(obs), exhibits a nonlinear dependence on reagent concentration, with K(I) = 19 microM and k(max) = 0.158 min(-)(1). Complete protection against inactivation is provided by tocopherol and tocopherol acetate, whereas glutathione derivatives, electrophilic substrate analogues, buffers, or nonsubstrate hydrophobic ligands have little effect on k(obs). These results indicate that TIA reacts as an affinity label of a distinguishable tocopherol binding site. Loss of activity occurs concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. Isolation of the labeled peptide from the tryptic digest shows that Tyr(79) is the only enzymic amino acid modified. The Y79F, Y79S, and Y79A mutant enzymes were generated, expressed, and purified. Changing Tyr(79) to Ser or Ala, but not Phe, renders the enzyme insensitive to inhibition by either tocopherol or tocopherol acetate as demonstrated by increases of at least 49-fold in K(I) values as compared to the wild-type enzyme. These results and examination of the crystal structure of GST pi suggest that tocopherols bind at a novel site, where an aromatic residue at position 79 is essential for binding.     

Affinity labeling of rat glutathione S-transferase isozyme 1-1 by 17beta -iodoacetoxy-estradiol-3-sulfate

Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Delta(5)-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17beta-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity. The rate constant for inactivation (k(obs)) exhibits a nonlinear dependence on reagent concentration with K(I) = 71 microm and k(max) = 0.0133 min(-1). Complete protection against inactivation is provided by 17beta-estradiol-3,17-disulfate, whereas Delta5-androstene-3,17-dione and S-methylglutathione have little effect on k(obs). These results indicate that 17beta-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Delta5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol 14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys(17) is the only enzymic amino acid modified. Covalent modification of Cys(17) by 17beta-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of the steroid analogue with subunit B. These results and examination of the crystal structure of the enzyme suggest that the interaction between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent and Arg(14) of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label.     

Affinity labeling of Escherichia coli DNA polymerase I by 5'-fluorosulfonylbenzoyladenosine. Identification of the domain essential for polymerization and Arg-682 as the site of reactivity

Preincubation of Escherichia coli DNA polymerase I (pol I) with 5'-fluorosulfonylbenzoyladenosine (5'-FSBA) results in an irreversible inactivation of DNA polymerase activity with concomitant covalent binding of 5'-FSBA to enzyme. pol I-associated 3'-5' exonuclease activity, however, remains unaffected. Kinetic studies of inactivation indicate that the degree of inactivation is directly proportional to the concentration of 5'-FSBA and increases linearly with time. The presence of the metal chelate form of dNTP substrates or template primer, but not the template or primer alone, protects the enzyme from inactivation by 5'-FSBA. A complete inactivation of polymerase activity occurs when 2 mol of 5'-FSBA are covalently linked to 1 mol of enzyme, suggesting two sites of modification. Tryptic peptide mapping of 5'-FSBA-treated enzyme revealed the presence of two distinct peptides containing the affinity label, confirming the presence of two reactive sites in the enzyme. However, we find that only one of the two sites is essential for the polymerase activity since, in the presence of substrate dNTP or template primer during preincubation of enzyme with 5'-FSBA, incorporation of the affinity label is reduced by only 1 mol. Peptide analysis of dNTP or template primer-protected enzyme further revealed that a peptide eluting at 35 min from the C-18 matrix was protected from the 5'-FSBA reaction. It is therefore concluded that this peptide contains the domain essential for polymerase activity. Staphylococcus aureus V-8 protease digestion, amino acid composition, and sequence analysis of this peptide revealed this domain to span residues 669 to 687 in the primary amino acid sequence of pol I, and arginine 682 was found to be the site of 5'-FSBA reactivity.     

Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase

A pterin analogue, 5-[(3-azido-6-nitrobenzylidene)amino]-2,6-diamino-4-pyrimidinone (ANBADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a Ki of 8.8 +/- 1.1 microM. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. Phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87 +/- 0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from [3H]ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with [3H]ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. The reactive sulfhydryl of phenylalanine hydroxylase associated with activation of the enzyme was also identified by labeling with the chromophoric label 5-(iodoacetamido)fluorescein [Parniak, M. A., & Kaufman, S. (1981) J. Biol. Chem. 256, 6876]. Labeling of the enzyme resulted in 1 mol of fluorescein bound per phenylalanine hydroxylase subunit and a concomitant activation of phenylalanine hydroxylase to 82% of the activity found with phenylalanine-activated enzyme. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.     

A conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2+-ATPase upon ATP binding to the catalytic site

Sarcoplasmic reticulum vesicles were modified with a fluorescent thiol reagent, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. One mol of readily reactive thiols per mol of the Ca2+-ATPase was labeled without a loss of the catalytic activity. The fluorescence of the label increased by 8% upon binding of Ca2+ to the high affinity sites of the enzyme. This fluorescence enhancement probably reflects a conformational change responsible for Ca2+-induced enzyme activation. Upon addition of ATP to the Ca2+-activated enzyme, the fluorescence decreased by 15%. This fluorescence drop and formation of the phosphoenzyme intermediate were determined under the same conditions with a stopped-flow apparatus and a rapid quenching system. The amplitude of the fluorescence drop thus determined was saturated with 3 microM ATP. This shows that the fluorescence drop was caused by ATP binding to the catalytic site. In contrast, the rate of the fluorescence drop was not saturated even with 50 microM ATP. The fluorescence drop coincided with phosphoenzyme formation at 0.5 or 3 microM ATP, but it became much faster than phosphoenzyme formation when the ATP concentration was raised to 100 microM. These results indicate that the ATP-induced fluorescence drop reflects a conformational change in the enzyme.ATP complex. The fluorescence drop was accompanied by a red spectrum shift, which suggests that the label was exposed to a more hydrophilic environment. The electrophoretic analysis of the tryptic digest of the labeled enzyme (10.9 kDa) showed that almost all of the label was located on the 5.2-kDa fragment which includes the carboxyl terminus and the putative ATP-binding domain. The sequencing of the two major labeled peptides, which were isolated from the thermolytic digest of the labeled enzyme, revealed that the labeled site in either of these peptides was Cys674. It seems likely that the label bound to this Cys674 could be involved in the observed fluorescence changes.     

Aspartyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate in the allosteric ADP site of pig heart NAD+-dependent isocitrate dehydrogenase

The nucleotide affinity label 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP) reacts covalently with pig heart NAD+-dependent isocitrate dehydrogenase with a limiting value of 75% inactivation and loss of ADP activation concomitant with incorporation of about 1 mol of reagent/mol of average enzyme subunit (Huang, Y.-C., Bailey, J. M., and Colman, R. F. (1986) J. Biol. Chem. 251, 14100-14107). Complete protection against the functional changes is provided by ADP + Mn2+, and reagent incorporation is decreased to about 0.5 mol/mol of average enzyme subunit. We have now identified the critical modified peptide by comparison of the peptides labeled by 2-BDB-TADP at pH 6.8 in the absence and presence of ADP + Mn2+. After removal of excess reagent, modified enzyme was treated with [3H]NaBH4 to reduce the keto groups of the reagent and introduce a radioactive tracer into the reagent which is covalently linked to the protein. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated using two successive high performance liquid chromatography steps: 1) 0.1% trifluoroacetic acid with an acetonitrile gradient; and 2) 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas phase sequencing gave the modified peptide Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln in which aspartic acid is the target of 2-BDB-TADP. Isolation of the corresponding tryptic peptide from unmodified enzyme yielded the sequence Leu-Gly-Asp-Gly-Leu-Phe-Leu-Gln-CmCys-CmCys-Lys. Isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma), separable by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The evidence indicates that the specific peptide labeled by 2-BDB-TADP, which is at or near the ADP site, can be derived from the gamma subunit.     

Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.     

Covalent modification of the inhibitor-binding site(s) of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-AMP-incorporated peptides

The photoaffinity inhibitor analog [2-3H]8-azido-AMP is specifically and covalently incorporated into Escherichia coli ADP-glucose synthetase. The reaction site(s) of [2-3H]8-azido-AMP with the enzyme was identified by reverse phase high performance liquid chromatography isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease-generated peptides containing the labeled analog. Three regions of modification, represented by six labeled peptides, accounted for over 85% of the covalently bound label. The major binding region of the azido analog, composed of residues 108-128, contained approximately 55% of the recovered covalently bound radioactivity. A single residue, Tyr-113, contained between 50 and 75% of the label found in the major binding region. This site is the same as the major binding region of the substrate site-specific probe, 8-azido-ADP-[14C]glucose (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). Conformational analysis of this region predicts that it is a part of a Rossmann fold, the supersecondary structure found in many adenine nucleotide-binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido-AMP were also identified by chemical characterization. One region, containing 20% of the covalently bound label, was composed of residues 11-68. This region contains Lys-38, the previously determined pyridoxal phosphate-modified allosteric activator site (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). The third minor region of modification, residues 222-254, contained approximately 15% of the covalently bound label. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure.     

Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate

NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.     

Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I

We have labeled the large fragment of Escherichia coli DNA polymerase I (Pol I) with pyridoxal 5'-phosphate, a substrate binding site directed reagent for DNA polymerases [Modak, M. J. (1976) Biochemistry 15, 3620-3626]. A covalent attachment of pyridoxal phosphate to Pol I results in the loss of substrate binding as well as the polymerase activity. The inactivation was found to be strictly dependent on the presence of a divalent metal ion. Four moles of pyridoxal phosphate was found to react per mole of the enzyme, while in the presence of substrate deoxynucleoside triphosphate only 3 mol of pyridoxal phosphate was bound. To identify the substrate-protected site on the enzyme, tryptic peptides from enzyme labeled with pyridoxal phosphate and tritiated borohydride, in the presence and absence of substrate, were resolved on a C-18 reverse-phase column. A single peptide containing the substrate-protected site was identified and further purified. The amino acid composition and sequence analysis of this peptide revealed it to span residues 756-775 in the primary acid sequence of Pol I. Lys-758 of this sequence was found to be the site of the pyridoxal phosphate reaction. It is therefore concluded that Lys-758 is the site of binding for the metal chelate form of nucleotide substrates in E. coli DNA polymerase I.     

Photoaffinity labeling of the catalytic site of prenyltransferase.

Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl pyrophosphate, and 3-azido-1-butyl pyrophosphate, have been synthesized as site-directed probes to label the catalytic site of prenyltransferase. Due to the relatively poor affinity of p-azidophenethyl pyrophosphate and 3-azido-1-butyl pyrophosphate for the enzyme, only o-azidophenethyl pyrophosphate (aryl azide) was utilized for photoaffinity labeling. This aryl azide has a UV absorption maximum at 250 nm. In the absence of activating light, binding studies demonstrate that the o-aryl azide competes for binding with both the natural substrates, isopentenyl pyrophosphate and geranyl pyrophosphate. More than 90% enzymatic activity is lost when enzyme is irradiated in the presence of the aryl azide as compared to irradiation in the absence of the azide, and the protein loses its capacity for substrate binding in direct proportion to photolabeling. A stoichiometry of 2 mol of affinity label covalently bound per mol of enzyme dimer was established with [1-3H]-o-azidophenethyl pyrophosphate. Since there are two catalytic sites per enzyme dimer, the o-aryl azide appears specifically to label the enzyme at its catalytic sites. Additional evidence that the reagent was specific for the catalytic site came from the observation that farnesyl pyrophosphate afforded complete protection against photoinactivation, while isopentenyl pyrophosphate provided partial protection. Gel isoelectric focusing verified this stoichiometry and indicated that the labeled enzyme has a more acidic isoelectric point than the native enzyme.     

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.     

Affinity labeling of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active-site cysteine

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 degrees C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-Ala-Cys-Thr-Glu-Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.     

Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label

The glutathione S-transferases are a family of dimeric enzymes that catalyze the reaction between GSH and a variety of electrophiles. Two closely related isozymes, referred to as YaYa and YcYc, were purified from rat liver. A radiolabeled azido derivative of glutathione (S-(p-azidophenacyl)[3H]glutathione) was prepared and used to label covalently the active site of the above two glutathione S-transferases. The noncovalently bound affinity label was a competitive inhibitor of glutathione S-transferase YaYa toward both 1-chloro-2,4-dinitrobenzene and GSH. The covalently labeled enzymes no longer bound to a GSH-affinity column, and covalent labeling was reduced in the presence of GSH and S-(dinitrophenyl)glutathione. These results suggest that the affinity label was binding at the active site. The covalently labeled enzymes were digested with trypsin, and the labeled peptides were purified by HPLC and then sequenced. A single-labeled peptide was identified in the tryptic digest of the YaYa isozyme, whereas two labeled peptides were present in the tryptic digest of YcYc. The Ya peptide sequence was identical with the published deduced sequence of amino acids between residues 212 and 218 and the sequences of the two peptides purified from Yc were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Ya peptide and the smaller peptide purified from Yc came from the same region of the Ya and Yc subunits. This common region and a second region of the Yc subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.     

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.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases.

The behavior of an array of fluorescent human alpha-thrombin derivatives in reporting binding of the fragment 2 domain of prothrombin was characterized as a representative application of the active-site-selective labeling approach to studies of blood coagulation proteinase regulatory interactions. An array of 16 thrombin derivatives was prepared by affinity labeling of the proteinase active site with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl or N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, followed by selective modification of the NH2OH-generated thiol group on the covalently incorporated inhibitors with each of eight thiol-reactive fluorescence probes. The changes in probe fluorescence intensity of the derivatives, signaling changes in the environment of the catalytic site associated with fragment 2 binding, appeared to be a unique and unpredictable function of the structure of the probe and the connecting peptide. These results demonstrated the utility of the labeling approach for overcoming the problem of not being able to predict which fluorescent label will provide the most useful proteinase derivative for investigating an interaction by enabling a greater variety of them to be prepared and screened for those with the most desirable properties. To determine whether the approach could be extended to other proteinases, the specificity of labeling with the fluorescence probe iodoacetamide, 5-(iodoacetamido)fluorescein, by use of the two thioester inhibitors was evaluated for several other blood coagulation proteinases and related trypsin-like enzymes. All of the proteinases were labeled in an active-site-selective manner. The combined results of quantitating the labeling reactions for the proteinase and inhibitor combinations studied thus far showed active-site-specific incorporation of 0.98 +/- 0.10 mol of inhibitor/mol of active sites and 0.92 +/- 0.11 mol of probe/mol of active sites, representing an overall greater than or equal to 93% site-specificity of labeling. These results demonstrated the broad applicability of the labeling approach for fluorescence studies of proteinases that differ greatly in their catalytic specificities.     

Identification of a lysine residue at a nucleotide binding site in the firefly luciferase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine

Firefly luciferase is 80-90% inactivated within 3 h upon incubation with the adenine nucleotide analogue p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). Although 4 mol of 14C-FSBA/mol of enzyme is irreversibly bound during inactivation, only 1 mol of 14C-FSBA appears to be specifically directed to an adenine nucleotide binding site on the enzyme. The other 3 mol of 14C-FSBA is bound to the protein nonspecifically. The major radioactive peptide in a tryptic digest os labeled luciferase was isolated and shown to have the following amino acid sequence: *Lys-Gly-Glx-Asx-Ser-Lys, where *Lys is the radioactive derivative of the lysine residue that was sulfonylated during the inactivation.