Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase

Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp(51). The D153E enzyme had an increased k(cat) in the presence of high concentrations of Mg(2+), along with a decreased Mg(2+) affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn(1) site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn(2+), dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.     

Chemical modification of the active site of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

The NADP-linked glutamate dehydrogenase (NADP-gluDH) purified from epimastigotes of the Tulahuén strain, Tul 2 stock, of Trypanosoma cruzi, was inhibited by Cibacron Blue FG3A, and inactivated by preincubation with phenylglyoxal or Woodward's Reagent K. The inhibition by Cibracron Blue FG3A, competitive towards NADPH with an apparent Ki of 20 microM, suggests that the enzyme presents the "dinucleotide fold" characteristic of most dehydrogenases and kinases. The inactivation of the NADP-gluDH by preincubation with phenylglyoxal, with a reaction order of 1, and the partial protection afforded by alpha-oxoglutarate, suggest the presence of one arginine residue in the active site of the enzyme, which might participate in the binding of alpha-oxoglutarate through interaction with one of the carboxyl groups of the substrate. The inactivation of the NADP-gluDH by preincubation with Woodward's Reagent K suggests the presence of a carboxyl group, from an aspartic or glutamic acid residue, at the active site, which might participate in the binding of the cationic substrate NH+4. The presence of NADPH during preincubation with the reagent increased the inactivation rate, which suggests that binding of the coenzyme increases the exposure of the reactive carboxyl group.     

Mapping the receptor-recognition site of human transforming growth factor-alpha.

The receptor-recognition site human transforming growth factor-alpha (TGF alpha), a 50-residue tricyclic peptide with three disulfide bonds, was mapped by a set of 46 peptide analogs consisting of linear, monocyclic, bicyclic, and tricyclic structures representing individual and overlapping subdomains of human TGF alpha. Linear overlapping fragments ranging from 7 to 18 residues and spanning the entire length of TGF alpha as well as monocyclic analogs with one disulfide linkage were found to be inactive in both receptor-binding and mitogenic assays. Bicyclic analogs with two disulfide linkage and representing either the amino or carboxyl two-thirds of TGF alpha showed low activity at 0.1-0.9 mM concentrations. Tricyclic analogs containing all three disulfide linkages but lacking either the amino or carboxyl terminal heptapeptide was, respectively, 3% and 0.1% as active as TGF alpha. These results show that determinants for the receptor binding cannot be represented by a short continuous fragment or a single subdomain, but are located on a discontinuous surface on a folded structure with disulfide restraints. Furthermore, these results when combined with our previous results which shows that the middle subdomain (second disulfide loop) is not involved in the receptor binding suggest that the receptor-binding residues are constituted of three fragments located at the first and third subdomains as well as the external carboxyl peptide.     

Study of the peptidasic site of cholinesterase: preliminary results

The peptidasic site of highly purified human plasma cholinesterase was investigated using active-site-directed inhibitors. Peptidase activity was assayed taking substance P as substrate. Inhibition by organophosphates indicated that the peptidasic site contained an active serine. The presence of essential histidine residues associated with serine was revealed by histidine modifications. Carboxyl group reagents showed that the active centre contained carboxyl groups in a non-polar environment. The removal of sialic acids did not alter peptidase activity. The peptidasic site of cholinesterase shared many properties with serine proteases sites and esteratic sites of cholinesterases. In addition, with the peptidasic site, as well as the esteratic site, there was always the possibility of 'aging' when inhibited by DFP or soman.     

Hypermodified nucleoside carboxyl group as a target site for specific tRNA modification

The free carboxyl group of hypermodified nucleosides N6-methyl-N6-(threoninocarbonyl)adenosine (mt6A37) and 3-(3-amino-3-carboxypropyl)uridine (acp3U20:1) in tRNAmMet (yellow lupine), and N6-(threoninocarbonyl)adenosine (t6A37) in tRNAiMet (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water-soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD-DEAE-cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono-modified (at mt6A37) and bis-modified (at mt6A37 and acp3U20:1) tRNAmMet, and for mono-modified (at t6A37) tRNAiMet by sequence analysis using 5'end 32P-labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.     

Probes of the catalytic site of cysteine dioxygenase

The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.     

Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase

Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp⁵¹. The D153E enzyme had an increased k_cat in the presence of high concentrations of Mg²⁺, along with a decreased Mg²⁺ affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn₁ site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn²⁺, dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.     

Clostridium pasteurianum glutamine synthetase mechanism. Evidence for active site tyrosine residues

Preliminary chemical modification studies indicated the presence of tyrosine, carboxyl, arginine, histidine and the absence of serine and sulfhydryl residues at or near the active site of Clostridium pasteurianum glutamine synthetase. The conditions for tyrosine modification with tetranitromethane were optimized. The inactivation kinetics follow pseudo-first-order kinetics with respect to enzyme and second order with respect to modifier per active site. There was no inactivation at pH 6.5 suggesting the absence of thiol oxidation. The synthetase and transferase reactions followed the same pattern of inactivation on enzyme modification and both were equally protected by glutamate plus ATP. Thus tyrosine residues are present at the active site of the enzyme and are essential for both transferase and synthetase activities.     

Cross-linking of the anticodon of P and A site bound tRNAs to the ribosome via aromatic azides of variable length: involvement of 16S rRNA at the A site

The topography of the ribosomal decoding site was explored by affinity labeling from the 5'-anticodon base, 5-(carboxymethoxy)uridine-34, of P or A site bound tRNA1Val. A nitrophenyl azide was attached to the carboxyl group of this nucleotide via side chains varying in length from 18 to 24 A. Binding of acetylvalyl-tRNA to the P site was codon dependent and that of valyl-tRNA to the A site was both codon and elongation factor Tu (EFTu) dependent. Cross-linking to both A and P sites was irradiation, probe, codon, and, in the case of the A site, EFTu dependent. Putative P-site cross-linked aminoacyl-tRNA was reactive with puromycin. The yield of cross-linking was little affected by placement of the tRNA at the A or P site but varied considerably with the length and structure of the probe side chain. When the distance from the pyrimidine C-5 atom to the azide group was 23 A, 42-45% cross-linking was obtained at each site, but when the distance was decreased to 18 A, only 7-12% was found. Placing an S-S bond in the center of the 23-A leash decreased the A-site yield to about half, while insertion of a CONH group decreased A-site cross-linking about 8-fold. P-site cross-linking was more sensitive to mercaptan quenching (50% at 0.5 mM) than was that at the A site (50% at greater than 2.0 mM) but both were partially shielded from solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the catalytic residues at the active site of human liver alpha-L-fucosidase

Kinetic studies and chemical modifications were performed on purified human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) in an attempt to identify the catalytic residues at the active site. Plots of log Vmax vs. pH (computer-fitted to a theoretical model) displayed two apparent pK values, of approx. 3.8 and 7.3. The temperature dependence of these pK values yielded heats of ionization of 3 and 0 kcal/mol from Van't Hoff plots for the lower and higher pK values, respectively. Reaction of alpha-L-fucosidase with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sodium p-(hydroxymercuri)benzoate resulted in complete inactivation of the enzyme. Other nonspecific inactivators had little or no effect on enzyme activity. These results suggest two carboxyl groups whose ionization state is important to activity, a non-active-site cysteine residue important to activity, and at least one active-site carboxyl group.     

Substrate specificity at the P1' site of Escherichia coli OmpT under denaturing conditions

Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg140-Arg141 and the modified substrates PRX substituted all 19 natural amino acids at the P1' site instead of Arg141. OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg140 except for the Arg140-Asp141, -Glu141, and -Pro141 pairs. In addition to Arg140 at the P1 site, similar results were obtained when Lys140 was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1' site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.     

Active site directed inactivators of mouse submaxillary renin.

The following active site directed inactivators for the pressor enzyme renin were synthesized: L-alpha-bromo-isocaproyl(BIC)-Leu-Val-Tyr-Ser-OH, L-BIC-Val-Tyr-Ser-OH, L-BIC-Leu-Val-OCH3, L-BIC-Leu-Val-OH, L-BIC-Val-Tyr-NH2, L-BIC-Val-Tyr-OCH3, L-BIC-Val-Tyr-OH, L-BIC-Leu-OCH3, L-BIC-Val-OCH3, and L-BIC-OCH3. The rate of inactivation of mouse submaxillary gland renin by these reagents was studied under a variety of conditions. L-alpha-Bromoisocaproyl-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH were the most efficient inactivators followed by L-alpha-bromoisocaproyl-Val-Tyr-NH2. The rates of inactivation by the first two peptides were strongly dependent on pH, being most efficient at low pH, least efficient at pH near 5.6, and becoming efficient again at neutral pH. The rate of the inactivation by L-alpha-bromoisocaproyl-Val-Tyr-NH2, in which the C-terminal carboxyl group is blocked, was only slightly dependent on pH. Complete inactivation was achieved by these three reagents. The inactivation was accompanied by incorporation of a stoichiometric quantity of the radiolabeled reagents. Based on these findings it was concluded that the inactivators reacted with a carboxyl group(s) in the active site of the renin molecule to form an esteric linkage. These data also suggest that a carcoxyl group(s) may constitute part of the catalytically essential functional groups in renin action. D-alpha-Bromoisocaproyl derivatives of the various peptides mentioned above were also prepared. These compounds were much less active than the L isomers indicating that the inactivation by the L-alpha-bromoisocaproyl peptides was a specific reaction.     

Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the binding site on cytochrome c1 for cytochrome c

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence for a carboxyl group at the active site

Ethanolamine ammonia-lyase is an adenosylcobalamin-dependent enzyme that catalyzes the rearrangement of ethanolamine and other vicinal amino alcohols to oxo-compounds and ammonia. Treatment of this enzyme with the sulfhydryl group-blocking reagent methyl methanethiosulfonate produces a species with diminished catalytic activity. When methyl methanethiosulfonate -treated ethanolamine ammonia-lyase was incubated with a carboxyl-blocking reagent consisting of glycine ethyl ester plus a water-soluble carbodiimide, the enzyme lost more than 80% of its residual activity, while at the same time glycine ethyl ester was incorporated into it at a stoichiometry of 6 mol/mol of enzyme. Both the loss of activity and the incorporation of glycine ethyl ester were prevented if ethanolamine was included in the glycine ethyl ester-containing incubation mixture. These results suggest that an active site carboxyl group plays a role in the mechanism of catalysis by ethanolamine ammonia-lyase, and that this carboxyl group is amidated when the enzyme is incubated with glycine ethyl ester plus carbodiimide.     

Phosphorylation of the beta subunit of casein kinase II in human A431 cells. Identification of the autophosphorylation site and a site phosphorylated by p34cdc2

To examine the phosphorylation of casein kinase II in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the alpha subunit or the beta subunit of the enzyme. When isolated from 32P-labeled cells, the beta subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the alpha subunit. In vitro, the beta subunit of purified bovine casein kinase II was autophosphorylated, also on serine residues. Cleavage of the beta subunit, that had been autophosphorylated in vitro, at tryptophan 9 and tryptophan 12 using N-chlorosuccinimide demonstrated that the autophosphorylation site is located near the amino terminus of the protein, most likely at serine 2 and serine 3. Two-dimensional maps of phosphopeptides generated by digestion of the beta subunit with endoproteinase Glu-C indicted that the majority of the phosphate that was incorporated into the protein in cells was at sites that were indistinguishable from the sites that were autophosphorylated in vitro. In addition to phosphorylation at the autophosphorylation site, the beta subunit is also phosphorylated at an additional site, serine 209, in intact cells. This residue, which is near the carboxyl terminus of the protein, can be phosphorylated in vitro by p34cdc2.     

A conserved steroid binding site in cytochrome C oxidase

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.     

Cosubstrate binding site of Pseudomonas sp. AK1 gamma-butyrobetaine hydroxylase. Interactions with structural analogs of alpha-ketoglutarate

Forty-one aromatic and aliphatic analogs of alpha-ketoglutarate were studied kinetically for their interaction with the alpha-ketoglutarate binding site of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp. AK1. Together, the compounds represent structural permutations probing the contribution of: 1) the C5 carboxyl group of alpha-ketoglutarate (domain I); 2) the C1-C2 keto acid moiety of alpha-ketoglutarate (domain II); 3) the distance between domains I and II; and 4) the spatial relationship of the two domains required for optimal interaction with the cosubstrate binding site. All compounds were competitive inhibitors for alpha-ketoglutarate (Km 0.018 mM). Functionally, two subsites of the cosubstrate binding site were evident: subsite I for polar interaction with the C5 carboxyl group, and subsite II, comprising of two distinct cis-oriented coordination sites of the catalytic ferrous ion which interact with the C1-C2 keto acid moiety. The most efficient inhibitors were pyridine 2,4-dicarboxylate (Ki 0.0002 mM) and 3,4-dihydroxybenzoate (Ki 0.0006 mM). Both compounds contain a carboxyl group and a chelating moiety corresponding to domains I and II of alpha-ketoglutarate, respectively. The fixed orientation of these groups in both analogs was used to assess intersubsite distance and spatial relationship required for optimal interaction with the cosubstrate binding site. Binding at subsite I and chelation at subsite II were indispensible for effective competitive inhibition. The distance between these two domains also helped determine whether attachment at the cosubstrate binding site would be catalytically productive. This was emphasized by the failure of either oxaloacetate or alpha-ketoadipinate to promote hydroxylation. Optimal interdomain distance, however, was not sufficient for cosubstrate utilization, as pyridine 2,4-dicarboxylate, with an interdomain distance identical to alpha-ketoglutarate in its staggered conformation, did not sustain hydroxylation. In the overall, these studies suggest that alpha-ketoglutarate utilization occurs in a ligand reaction at the active site ferrous ion of gamma-butyrobetaine hydroxylase. This is of particular interest since the delineated stereochemical mode of oxidative decarboxylation could generate the reactive oxo-iron species that was shown experimentally to promote gamma-butyrobetaine hydroxylation by an abstraction-recombination mechanism (Blanchard, J. S., and Englard, S. (1983) Biochemistry 22, 5922-5928; Englard, S., Blanchard, J. S., and Midelfort, C. F. (1985) Biochemistry 24, 1110-1116).     

Identification of a site for carboxyl methylation in human alpha-globin

The human erythrocyte protein carboxyl methyltransferase modifies unusual protein D-aspartyl and L-isoaspartyl residues which arise spontaneously from internal rearrangements accompanying asparaginyl deamidation and aspartyl isomerization. A site of methylation associated with alpha-globin in intact cells has been identified by peptide mapping of radiolabeled globin isolated from human erythrocytes previously incubated with L-[methyl-3H]methionine. The site is located in a Staphylococcus V8 peptide containing residues 1-30 of alpha-globin. Two potential sources of methylation sites are present in this sequence at Asp-t and Asn-9.     

Trypsin-sensitive neutralization site on VP1 of Theiler''s murine encephalomyelitis viruses.

We generated Theiler's murine encephalomyelitis virus mutants resistant to several neutralizing monoclonal antibodies (MAbs) having their epitopes near a trypsin cleavage site of VP1. Neutralization and Western blot (immunoblot) studies suggest that two of the MAbs have identical epitopes that partly overlap the epitope of a third MAb. Sequencing of RNA of the mutants localized the epitopes to a site near the carboxyl end of VP1. The limited diversity of nucleotide changes seen in the mutants and the immunodominance of the site suggest that the carboxyl end of VP1 may have an important function.