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PubMed1997

Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis.

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.

Amino Acids↗

PubMed2003

Aquifex aeolicus tRNA (Gm18) methyltransferase has unique substrate specificity. TRNA recognition mechanism of the enzyme.

Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates.

Amino Acid Sequence↗

PubMed2004

Transmembrane domains I and II of the gamma-aminobutyric acid transporter GAT-4 contain molecular determinants of substrate specificity.

The sodium- and chloride-dependent GABA transporters GABA transporter (GAT) 1 to 4 in the central nervous system enable efficient synaptic transmission by removing the neurotransmitter from the cleft. Taurine interacts only weakly with the GABA transporter GAT-4 (IC50 approximately 1.6 mM). Glutamate-61 is located in the conserved transmembrane domain I of GAT-4, whereas in the related taurine-transporter taurine transporter (TAUT), glycine occupies the equivalent position. [3H]GABA uptake by the GAT-4 E61G mutant becomes markedly more sensitive to inhibition by taurine (IC50 approximately 0.26 mM). Replacement of cysteine-94, located in the conserved transmembrane domain II of GAT-4, to its TAUT counterpart serine, results only in a modest increase in the ability of taurine to inhibit GABA uptake. However, introduction of glycine at this position decreases the IC50 for taurine by approximately 8-fold (IC50 approximately 0.20 mM). The inhibitory potency of taurine is inversely correlated with the volume of the side chain of the amino acid residue introduced at positions 61 and 94. It is striking that the IC50 for taurine of the E61G/C94G double mutant is decreased by approximately 35-fold (IC50 approximately 0.05 mM), and this inhibition of GABA transport is competitive. Changes in the inhibitory potency of the mutants described are also observed with beta-ala-nine and GABA, although they are much less pronounced. Our results suggest that determinants on transmembrane domains I and II can influence the specificity of the substrate binding pocket. The size of the side chain at positions 61 and 94 seems to determine the ability of substrate and substrate analogs to interact with the transporter.

Animals↗

PubMed2003

Regulation and substrate specificity of the SR protein kinase Clk/Sty.

SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

Blotting, Western↗

PubMed1989

[Substrate specificity of multiple forms of human alpha-D-galactosidase and alpha-D-fucosidase].

It was shown that human alpha-D-galactosidase is represented by multiple forms, only one of which can also split alpha-D-fucoside. Fabry's disease was found to be associated not only with the deficiency of the alpha-D-galactosidase total activity but also with the deficiency of the alpha-D-fucosidase activity. The decrease in the alpha-D-galactosidase activity is due to the lack of two enzyme forms, while the profile of alpha-D-fucosidase multiple forms during isoelectric focusing of human enzyme preparations is modified very little in comparison with the normal one. The deficiency of both enzymes was expressed in most degree in leukocytes as compared to other tissues. The residual activities of alpha-D-galactosidase and alpha-D-fucosidase in leukocytes were equal to 3.5 and 21%, respectively. Since the decrease in the alpha-D-fucosidase activity was not so noticeable as in the alpha-D-galactosidase activity, it may be expected that the determination of the alpha-D-fucosidase activity can no longer be regarded as a reliable test for the diagnosis of Fabry's disease. The data obtained suggest that alpha-D-galactoside and alpha-D-fucoside are split by the same enzyme, the multiple forms of which are characterized by selective specificity towards these substrates.

Fabry Disease↗

PubMed1994

Classification of some alpha-glucosidases and alpha-xylosidases on the basis of substrate specificity.

Three alpha-glucosidases which passed under the names of transglucosidase (from Aspergillus niger), maltase (from Brewers yeast), and isomaltase (from Bakers yeast) for reasons of their substrate specificities and transfer actions, were purified to electrophoretically pure states. These purified alpha-glucosidases were made uniform in the hydrolyzing activities using p-nitrophenyl alpha-glucopyranoside (alpha-p-NPG) and were reacted with p-nitrophenyl alpha-xylopyranoside (alpha-p-NPX) or isoprimeverose (xylopyranosyl-alpha-1,6-glucopyranose), which are typical substrates of alpha-xylosidase. Only Asp. niger alpha-glucosidase among them hydrolyzed alpha-p-NPX and isoprimeverose. Further the substrate specificities of three alpha-glucosidases and two alpha-xylosidases (I and II from Asp. flavus MO-5) were investigated on maltose, isomaltose, alpha-p-NPG, isoprimeverose, and alpha-p-NPX in detail, and kinetic parameters [Km, Vmax, and molecular activity (Ko)] were estimated and compared with each other. In the comparison of kinetic parameters, Asp. niger alpha-glucosidase showed a broad specificity, that is, containing isoprimeverose in addition to maltose, isomaltose, and alpha-p-NPG. Though this enzyme barely hydrolyzed alpha-p-NPX too, the velocity was very slow. Though both yeast alpha-glucosidases barely hydrolyzed alpha-p-NPX or isoprimeverose too, these substrates were not good for yeast enzymes. On the other hand, two alpha-xylosidases showed narrow specificities, such that the substrates except for alpha-p-NPX and isoprimeverose were not hydrolyzed at all. The action on isoprimerose by Asp. niger alpha-glucosidase was completely the same as that on isomaltose at optimum pH, optimum temperature, inhibition pattern of hydrolyzing activity by 1-deoxynojirimycin, and transfer action pattern. Accordingly, we interpret these results as indicating that the hydrolyzations of isomaltose and isoprimeverose by Asp. niger alpha-glucosidase were catalyzed at the same active site. Asp. niger enzyme that has both alpha-glucosidase activity and alpha-xylosidase activity was shown to be classified in a middle position between alpha-glucosidase and alpha-xylosidase.

Aspergillus niger↗

PubMed1995

[Preparation of specific substrates for glycopeptidamidase A and kinetics of their enzymatic hydrolysis].

Glycopeptides were obtained by the pepsin (glycononapeptide) and trypsin (glycohexapeptide) hydrolysis of ovalbumin. A method (HPLC) of the glycopeptidamidase activity determination with a fluorescent-labelled (Dns) glycopeptides is described, and kinetic constants of hydrolysis of specific substrates by glycopeptidamidase A are determined. Dns(O-Dns)Tyr-Asn(CHO)-Leu-Thr-Ser-Val is shown to be a specific substrate of the glycopeptidamidase A.

Amidohydrolases↗

PubMed1983

Phosphatidylinositol transfer protein from bovine brain. Substrate specificity and membrane binding properties.

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.

Animals↗

PubMed1986

Analysis of sialidase and N-acetylneuraminate pyruvate-lyase substrate specificity by high-performance liquid chromatography.

A rapid and sensitive assay by high-performance liquid chromatography for determination of the activity and substrate specificity of sialidase (EC 3.2.1.18) and N-acetylneuraminate lyase (EC 4.1.3.3) is described. Sialic acids were separated on a strong anion-exchange resin using 0.75 mM sodium sulfate as elution medium. This method allows the determination of a minimum amount of 200 pg (0.6 pmol) of sialic acid. Usually the enzyme mixtures were directly applied to the column without prior purification of substrates and products. The action of sialidase was studied either by the decrease of sialyllactose concentration or by the amount of sialic acid liberated. The relative hydrolysis rates of N-acetylneuraminyl-alpha(2-3)-lactose, N-glycolylneuraminyl-alpha(2-3)-lactose, N-acetylneuraminyl-alpha(2-6)-lactose, N-acetyl-9-O-acetylneuraminyl-alpha(2-3)-lactose, and N-acetyl-4-O-acetylneuraminyl-alpha(2-3)-lactose by Vibrio cholerae sialidase were 100, 88, 25, 12, and 0, respectively. The activity of N-acetylneuraminate lyase from Clostridium perfringens was determined by measuring the rate of disappearance of sialic acids or the formation of acylmannosamines, which is possible in the same chromatogram. Relative cleavage rates of N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, and N-acetyl-4-O-acetylneuraminic acid were found to be 100, 67, 24, 3, and 0, respectively. Comparison of the substrate specificities shows that substituents on the neuraminic acid molecule influence the reactions of both enzymes in a similar way.

Chromatography, High Pressure Liquid↗

PubMed1999

Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.

The two multifunctional cytochrome P450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes aromatic oximes efficiently, whereas aliphatic oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metabolism of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0. 3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temperature. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compounds which do not serve as substrates formed Type I substrate binding spectra with the two P450 enzymes.

Catalysis↗

PubMed1997

Substrate specificity of the hepatitis C virus serine protease NS3.

The substrate specificity of a purified protein encompassing the hepatitis C virus NS3 serine protease domain was investigated by introducing systematic modifications, including non-natural amino acids, into substrate peptides derived from the NS4A/NS4B cleavage site. Kinetic parameters were determined in the absence and presence of a peptide mimicking the protease co-factor NS4A (Pep4A). Based on this study we draw the following conclusions: (i) the NS3 protease domain has an absolute requirement for a small residue in the P1 position of substrates, thereby confirming previous modelling predictions. (ii) Optimization of the P1 binding site occupancy primarily influences transition state binding, whereas the occupancy of distal binding sites is a determinant for both ground state and transition state binding. (iii) Optimized contacts at distal binding sites may contribute synergistically to cleavage efficiency.

Alanine↗

PubMed2000

Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli.

In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes encoding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. The fatty acid beta-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad mutants harboring plasmid pBHR68, the R. eutropha PHA synthase led to accumulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively. Coexpression of phaC1 from P. aeruginosa indicated and confirmed the provision of PHA precursor via the beta-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data strongly suggested that R. eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD.

Acyltransferases↗

PubMed2000

The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility.

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition. In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation leads to a reduction in Pdr5p-mediated azole and rhodamine 6G resistance. Like S1360F, the T1364F and T1364A mutants were nearly non-responsive to FK506 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p-S1360A is hypersensitive to FK506 inhibition when compared with either wild-type Pdr5p or the non-responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents. This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.

ATP-Binding Cassette Transporters↗

PubMed1998

Evolution of substrate specificities in the P-type ATPase superfamily.

P-type ATPases make up a large superfamily of ATP-driven pumps involved in the transmembrane transport of charged substrates. We have performed an analysis of conserved core sequences in 159 P-type ATPases. The various ATPases group together in five major branches according to substrate specificity, and not according to the evolutionary relationship of the parental species, indicating that invention of new substrate specificities is accompanied by abrupt changes in the rate of sequence evolution. A hitherto-unrecognized family of P-type ATPases has been identified that is expected to be represented in all the major phyla of eukarya.

Adenosine Triphosphatases↗

PubMed2005

The C-terminal domain of the nucleotide-binding domain protein Wzt determines substrate specificity in the ATP-binding cassette transporter for the lipopolysaccharide O-antigens in Escherichia coli serotypes O8 and O9a.

The polymannan O-antigenic polysaccharides (O-PSs) of Escherichia coli O8 and O9a are synthesized via an ATP-binding cassette (ABC) transporter-dependent pathway. The group 2 capsular polysaccharides of E. coli serve as prototypes for polysaccharide synthesis and export via this pathway. Here, we show that there are some fundamental differences between the ABC transporter-dependent pathway for O-PS biosynthesis and the capsular polysaccharide paradigm. In the capsule system, mutants lacking the ABC transporter are viable, and membranes isolated from these strains are no longer able to synthesize polymer using an endogenous acceptor. In contrast, E. coli strains carrying mutations in the membrane component (Wzm) and/or the nucleotide-binding component (Wzt) of the O8 and O9a polymannan transporters are nonviable under conditions permissive to O-PS biosynthesis and take on an aberrant elongated cell morphology. Whereas the ABC transporters for capsular polysaccharides with different structures are functionally interchangeable, the O8 and O9a exporters are specific for their cognate polymannan substrates. The E. coli O8 and O9a Wzt proteins contain a C-terminal domain not present in the corresponding nucleotide-binding protein (KpsT) from the capsule exporter. Whereas the Wzm components are functionally interchangeable, albeit with reduced efficiency, the Wzt components are not, indicating a specific role for Wzt in substrate specificity. Chimeric Wzt proteins were constructed in order to localize the region involved in substrate specificity to the C-terminal domain.

ATP-Binding Cassette Transporters↗

PubMed1993

Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes.

Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub-groups: the Ca(2+)-dependent (classical) PKC isoenzymes alpha, beta I, beta II and gamma, and the Ca(2+)-independent neoPKC isoenzymes delta, epsilon, zeta, eta and theta. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction. In this study, we report the cloning of a human PKC-zeta cDNA, its expression in recombinant baculovirus-infected insect cells and the partial purification of the PKC-zeta isoenzyme. In comparison to highly purified human PKC alpha, a representative of the classical PKC subgroup, purified PKC zeta was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC alpha, PKC zeta exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of gamma-linolenic acid and phosphatidylserine slightly enhance PKC zeta activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC alpha phosphorylates a PKC-alpha pseudosubstrate-derived peptide, an epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-alpha-derived peptide. However, arachidonic acid greatly diminishes PKC-alpha activity towards the epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein, but enhances PKC-zeta activity towards the PKC-alpha-derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC zeta, this finding is strengthened by the fact that the epidermal growth factor receptor-derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC zeta, in contrast to PKC alpha, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) of activation and the substrate specificity of PKC zeta are discussed.

Amino Acid Sequence↗

PubMed1999

Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a dimeric, bacterial enzyme that detoxifies many organophosphorus neurotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 are located near the bimetallic active site present in each monomer. It has been proposed that these residues influence catalysis by interacting with active site residues and the substrate in the binding pocket. We replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single-site-modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site (four per dimer), all three of these altered enzymes possessed only two metals per dimer while retaining considerable enzymatic activity for the preferred phosphotriester (P-O bond) substrate, paraoxon (5-100% kcat). The three altered enzymes achieved a 2-30-fold increase in substrate specificity (kcat/Km) for demeton S (P-S bond), an analogue for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (P-F bond) was substantially decreased for each of these enzymes. In addition, H257L and H254R/H257L showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analogue for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and it is suggested that changes in metal requirements may affect these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site, while simultaneously decreasing the catalytic efficiency for smaller substrates.

Aryldialkylphosphatase↗

PubMed1990

Substrate specificities of the peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes.

Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.

Amino Acid Isomerases↗