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PubMed1988

Substrate specificity of rat liver glutathione S-transferase isoenzymes for a series of glutathione analogues, modified at the gamma-glutamyl moiety.

The substrate specificity of purified rat liver glutathione S-transferases (GSTs) for a series of gamma-glutamyl-modified GSH analogues was investigated. GST isoenzyme 3-3 catalysed the conjugation of 1-chloro-2,4-dinitrobenzene with six out of the nine analogues. alpha-L-Glu-L-Cys-Gly and alpha-D-Glu-L-Cys-Gly showed catalytic efficiencies of 40% and 130% that of GSH respectively. The GSH analogue with an alpha-D-glutamyl moiety appeared to be a highly isoenzyme-3-3-specific co-substrate: kcat./Km with GST isoenzyme 4-4 was only about 5% that with GST isoenzyme 3-3, and no enzymic activity was detectable with GST isoenzymes 1-1 and 2-2. GST isoenzyme 4-4 showed some resemblance to GST 3-3: five out of nine co-substrate analogues were accepted by this second isoenzyme of the Mu multigene family. Isoenzymes 1-1 and 2-2, of the Alpha multigene family, accepted only two alternative co-substrates, which indicates that their GSH-binding site is much more specific.

Animals↗

PubMed2002

Chemoenzymatic synthesis of a bifunctionalized cellohexaoside as a specific substrate for the sensitive assay of cellulase by fluorescence quenching.

A new bifunctionalized cellohexaose derivative was synthesized as a specific substrate for continuous assay of cellulases by resonance energy transfer. This cellohexaoside has a naphthalene moiety (EDANS) as a fluorescent energy donor at the reducing end and a 4-(4'-dimethylaminobenzeneazo)-benzene derivative as an acceptor chromophore at the non-reducing end. The key steps for the preparation of the target molecule involved transglycosylation reactions of cellobiosyl and cellotetraosyl fluoride donors onto cellobiosyl acceptors catalysed by the E197A mutant of cellulase Cel7B from Humicola insolens. Upon digestion with various cellulases, the energy transfer was disrupted and an increase of fluorescence was observed.

Cellulase↗

PubMed1989

Substrate specificity and variables affecting efficiency of mammalian flavin adenine dinucleotide synthetase.

Substrate specificity and product inhibition have been evaluated by using purified rat liver FAD synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2), obtained by an improved purification protocol with optimized flavin affinity chromatography. FMN analogues studied fall into three general classifications: those with substitution on the pyrimidinoid ring and nitrogen replacement, those with substitution on the benzenoid ring, and those with N(10) side chain modifications. Substitutions on the pyrimidinoid ring and replacement of nitrogens have the greatest influence on binding to enzyme and FAD formation. When the hydrogen-bonding capacity of the NH group at position 3 is blocked or removed by substitution, such FMN analogues do not act as substrates or inhibitors of the enzyme. Substitutions on the benzenoid ring by small groups seem to be tolerated, while larger groups inhibit binding. Length of the N(10) side chain is optimal with five carbons and has greatest affinity for the natural ribityl side chain. Affinity matrices show similar binding characteristics in that the N(3)-(carboxymethyl)riboflavin-agarose does not bind enzyme, while agaroses linked to the flavin N(10) side chain provide varying degrees of purification. The C = O group at position 2, the NH group at position 3, and a five-carbon side chain at the N(10) position seem to be most crucial for flavin substrate binding to enzyme. Nucleoside triphosphates other than ATP do not act as substrates or inhibitors when sufficient Mg2+ is present. Products of the reaction, FAD and PPi, act as inhibitors against both ATP and FMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1998

Species differences in substrate specificity of lipoprotein lipase purified from chickens and rats.

Kinetic parameters of chicken and rat lipoprotein lipase (LPL) were determined in the incubation in vitro with various monoacid triacylglycerol emulsion and plasma lipoproteins. In rat- and chicken-LPL there is an inverse relationship between the hydrolytic rate by both LPL and the increased acyl-chain unsaturation of monoacid triacylglycerol; C18:1>C18:2>C18:3. The rat LPL catalyzed hydrolysis of saturated monoacid triaclyglycerol increased with an increase of chain length as C16>C14>C12, whereas in chicken LPL hydrolytic rate of C12 was higher than C14 and C16 triaclyglycerol. Vmax of rat- and chicken-LPL for chylomicron and VLDL were higher but apparent Km for those were lower than other lipoproteins. In chicken, Vmax and apparent Km of LPL for VLDL were almost the same as those for chylomicron, whereas in rat, Vmax of LPL for VLDL was twice that of chylomicron with the same apparent Km. The chicken and rat VLDL with different particle size prepared by Bio-Gel A50 gel chromatography were similarly hydrolyzed by LPL, while the hydrolysis of small chicken-chylomicron particles was inclined to be higher than that of the large particles. These results show species differences between chickens and rats in the substrate specificity of LPL.

Animals↗

PubMed1992

Substrate specificity of cholesterol oxidase from Streptomyces cinnamomeus--a monolayer study.

The substrate specificity of cholesterol oxidase from Streptomyces cinnamomeus was examined in oriented sterol monolayers at the air/water interface. Of the cholesterol analogues with structural alterations in the A- or B-ring that were examined, it was observed that 5 alpha-cholestan-3 beta-ol was oxidized almost as fast as cholesterol itself. When the delta-5 double bond in cholesterol was instead at the delta-4 position, the oxidation rate became 3.2-fold slower. A similar reduction in the average oxidation rate was observed when the delta-5 double bond in cholesterol was instead at the delta-7 position (5 alpha-cholest-7-en-3 beta- ol). 5,7-Cholestadien-3 beta-ol was oxidized 5.1-fold slower compared to cholesterol, whereas 3 beta-hydroxy-5-cholesten-7-one and 5 beta-cholestan-3 beta-ol were not substrates of the enzyme (also verified from the lack of H2O2-production). With C(17) side chain analogues of cholesterol, it was observed that the complete lack of the C(17) side chain (5-androsten-3 beta-ol), or the insertion of an unsaturation at delta-24 (desmosterol), or even an ethyl group at C(24)(24b-ethyl-5,22- cholestadien-3 beta-ol) had no appreciable effects on sterol oxidation rate, implying that the enzyme did not recognize the side chain in oriented sterol monolayers. This study has shown that the sterol monolayer system is a good technique to examine sterol/cholesterol oxidase interactions, since both the orientation of the substrate molecules, and the quality of the interface can be mastered.

Cholesterol↗

PubMed2004

Animal deoxyribonucleoside kinases: 'forward' and 'retrograde' evolution of their substrate specificity.

Deoxyribonucleoside kinases, which catalyse the phosphorylation of deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate specificity. Recent studies suggest that in the animal lineage one of the progenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC 2.7.1.145), which subsequently, through remodelling of a limited number of amino acid residues, gained a broad substrate specificity.

Amino Acid Sequence↗

PubMed1980

Synthesis and analytical use of 3-carboxypropionyl-alanyl-alanyl-valine-4-nitroanilide: a specific substrate for human leukocyte elastase.

A simple synthesis is described for 3-carboxypropionyl-Ala-Ala-Val-4-nitroanilide, a convenient and very specific substrate for human leukocyte elastase (Km = 1.0mM, kcat = 8.7 s-1). The substrate does not undergo appreciable spontaneous hydrolysis. It is not cleaved by trypsin or chymotrypsin and only rather slowly by porcine pancreatic elastase (Km = 9.1mM, kcat = 1.4 s-1).

Humans↗

PubMed1991

The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities.

Cloning and expression of the homologous domains of the receptor-like tyrosine phosphatase HPTP alpha shows that both domain 1 (D1) and domain 2 (D2) are enzymatically active. The two domains display different substrate specificities with D1 preferentially dephosphorylating MBP approximately RR-src greater than PNPP while D2 favours PNPP much much greater than RR-src and is inactive towards MBP. Each domain has lower activity than an expressed protein containing both domains. Analysis of chimaeric D1/2 proteins suggests that no particular region of D2 is responsible for the low activity of D2 on RR-src and that the specificity differences of D1 and D2 reflect overall sequence dissimilarities. Activities of D1 and D2 are inhibited by zinc, vanadate and EDTA and differentially susceptible to inhibition by heparin and poly(Glu4:Tyr1). Unusually, the activity of the protein containing both domains is stimulated by these polyanions. Regions amino-terminal to each domain are important for catalysis since deletion of these sequences abolishes phosphatase activity. Activity of the double domain polypeptide was also lost upon deletion of the sequence amino-terminal to D1, indicating that inactivation of D1 may suppress D2 activity. Differences in substrate specificity and responses to effectors and the interdependence between the two domains are likely important properties in the function of this PTPase in signal transduction.

Amino Acid Sequence↗

PubMed1996

Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes.

The D-alanine:D-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesize D-alanyl-D-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesize D-alanyl-D-lactate or d-alanyl-d-serine. The sequence of internal fragments of eight structural d-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (D-alanine:D-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and DdlA and DdlB from enteric bacteria and Haemophilus influenzae constituted separate clusters.

Amino Acid Sequence↗

PubMed2001

Use of indirect site-directed mutagenesis to alter the substrate specificity of methylamine dehydrogenase.

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone-dependent enzyme that catalyzes the oxidative deamination of primary amines. Native MADH exhibits a strong preference for methylamine over longer carbon chain amines. Residue alphaPhe(55) controls this substrate specificity. When alphaPhe(55) is replaced with Ala, this preference is reversed with alphaF55A MADH preferring long-chain amines with at least seven carbons (Zhu, Z., Sun, D., and Davidson, V. L. (2000) Biochemistry 39, 11184-11186). To further modulate the substrate specificity of MADH, the side-chain of alphaPhe(55) was repositioned by site-directed mutagenesis of residue betaIle(107). This residue makes close contact with alphaPhe(55) and restricts its movement. betaI107V MADH exhibits a strong preference for propylamine, and betaI107N MADH exhibits a preference for 1-aminopentane. Thus, it has been possible to create forms of MADH that exhibit a preference for amines with carbon chain lengths of one, three, five, or seven carbons. The ability to discriminate between amines of different chain length was essentially abolished by an alphaF55I mutation. Molecular modeling studies with the known crystal structure are described that provide an explanation for these results. These results provide an example of a design-based approach to protein engineering in which site-directed mutagenesis on one residue can be used to reposition another residue to specifically alter enzyme specificity.

Base Sequence↗

PubMed1989

New substrate specificity of modified porcine pancreatic alpha-amylase.

Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.

Amylases↗

PubMed1992

Substrate specificity of diazepam-insensitive cerebellar [3H]Ro 15-4513 binding sites.

[3H]Ro 15-4513, a negative modulator at the benzodiazepine receptor, binds with high affinity to all known benzodiazepine binding sites associated with the GABAA receptors. The present experiments surveyed a number of benzodiazepine receptor ligands with different chemical structures and different intrinsic pharmacological profiles for their ability to displace [3H]Ro 15-4513 binding in rat cerebellar membranes. The latter have been shown to possess two types of binding sites, one sensitive to positive modulators such as diazepam and the other insensitive to them (diazepam-insensitive). Whereas the full positive modulators from the benzodiazepine, beta-carboline, and quinoline classes did not displace binding at the diazepam-insensitive sites, the partial positive modulators' affinities for these sites varied much more, some being very potent and others having no affinity at all. All the neutral antagonists tested displaced binding at both types of binding sites, while some of the negative modulators were apparently not potent at the diazepam-insensitive sites. In an alcohol-sensitive rat line (ANT, alcohol non-tolerant), with an enhanced affinity for positive modulators at the diazepam-insensitive sites, these sites also exhibited enhanced affinity for some of the partial positive and negative modulators. The results suggest that the cerebellar diazepam-insensitive [3H]Ro 15-4513 binding sites have unique substrate specificity. It remains to be established which behaviours are affected by activation or inhibition of these receptors that are characterized by their insensitivity to benzodiazepine-positive modulators.

Animals↗

PubMed1994

Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y.

Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.

Amino Acid Sequence↗

PubMed2003

Molecular cloning and heterologous expression in E. coli of cytochrome P45017alpha. Comparison of structural and functional properties of substrate-specific cytochromes P450 from different species.

To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017alpha from species of the Bovidae family (sheep, goat, and bison), which catalyze 17alpha-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C(19)-steroids. Recombinant cytochromes P45017alpha were expressed in E. coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography. Highly purified cytochromes P45017alpha were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17alpha-hydroxyprogesterone, and 17alpha-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC. It is shown that each form of cytochrome P45017alpha is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b(5) in the reaction mixture. The analysis of the activity of the known forms of cytochrome P45017alpha in view of the data obtained in the present work allows the division of known cytochromes P45017alpha into three main group: group A (pig, hamster, rat), cytochromes P45017alpha catalyze the reaction of 17alpha-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17alpha-hydroxyprogesterone and 17alpha-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017alpha, which have no or have insignificant 17,20-lyase activity in relation to 17alpha-hydroxyprogesterone; group C (guinea pig), cytochrome P45017alpha which either has no or has insignificant 17,20-lyase activity on transformation 17alpha-hydroxypregnenolone to dehydroepiandrosterone.

Amino Acid Sequence↗

PubMed2000

[Determination of substrate specificity of fucosyltransferase from rat brain using synthetic acceptors].

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Gal beta 1-4GlcNAc beta 1-R fragment were subjected to enzymic fucosylation. The isomer with a 1-3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated whereas Fuc alpha 1-2Gal beta 1-4GlcNAc displayed the same substrate properties as Gal beta 1-4GlcNAc. FT from cerebellum and forebrain was shown to have the specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from forebrain, the specificity profiles being similar.

Animals↗

PubMed2000

Substrate specificity of regiospecific desaturation of aliphatic compounds by a mutant Rhodococcus strain.

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.

Fatty Acids↗

PubMed2000

Human flavin-containing monooxygenase: substrate specificity and role in drug metabolism.

The human flavin-containing monooxygenase (FMO3) is a prominent enzyme system that converts nucleophilic heteroatom-containing chemicals, drugs and xenobiotics to more polar materials that are more efficiently excreted in the urine. The substrate specificity for FMO 3 is distinct from that of FMO1. Human FMO3 N-oxygenates primary, secondary and tertiary amines whereas human FMO1 is only highly efficient at N-oxygenating tertiary amines. Both human FMO1 and FMO3 S-oxygenate a number of nucleophilic sulfur-containing substrates and in some cases, does so with great stereoselectivity. Human FMO3 is sensitive to steric features of the substrate and aliphatic amines with linkages between the nitrogen atom and a large aromatic group such as a phenothiazine of at least five carbons are N-oxygenated significantly more efficiently than those substrates with two or three carbons. For amines with smaller aromatic substituents such as phenethylamines, often these compounds are efficiently N-oxygenated by human FMO3. Currently, the most promising non-invasive probe of in vivo human FMO3 functional activity is the formation of trimethylamine N-oxide from trimethylamine that comes from dietary choline. (S)-Nicotine N-1'-oxide formation can also be used as a highly stereoselective probe of human FMO3 function for adult humans that smoke cigarettes. Finally, cimetidine S-oxygenation or ranitidine N-oxidation can also be used as a functional probe of human FMO3. With the recent observation of human FMO3 genetic polymorphism and poor metabolism phenotype in certain human populations, variant human FMO3 may contribute to adverse drug reactions or exaggerated clinical response to certain medications. Knowledge of the substrate specificity for human FMO3 may aid in the future design of more efficacious and less toxic drugs.

Animals↗

PubMed1998

Di- and oligosaccharide substrate specificities and subsite binding energies of pig intestinal glucoamylase-maltase.

The substrate specificity of pig intestinal glucoamylase-maltase was investigated. The alpha-1, beta-2-glycosidic bond of the disaccharide sucrose was not hydrolyzed. Various substrates with alpha-1,4-glycosidic bonds (maltose, maltooligosaccharides) were hydrolyzed with high maximal reaction velocities. Reduction lowered the rate of hydrolysis drastically: k'0 decreases from 75 s-1 for maltose to 3 s-1 for maltitol while the K(m) value increases from 4.2 to 50 mM. Leucrose with alpha-1,5-glycosidic bond was hydrolyzed with a k'0 value of 8 s-1 and a K(m) value of 74 mM. Disaccharides with alpha-1,6-glycosidic bonds were hydrolyzed with extremely low rates: for isomaltose and isomaltulose k'0 values of 5 and 3 s-1, respectively, and K(m) values of 90 and 42 mM, respectively, were observed. Again reduction lowers the k'0 values: The corresponding disaccharide alcohols alpha-D-glucopyranosyl-1,6-sorbitol and alpha-D-glucopyranosyl-1,6-mannitol, like isomaltooligosaccharides, were not hydrolyzed. Regarding the conformation of sucrose, leucrose, and maltose previously determined by molecular dynamics simulations, a reasonable explanation for the different rates of hydrolysis could be postulated. Based on the enzyme kinetic parameters for the series of maltooligosaccharides, subsite affinities (A1) according to the subsite theory were calculated as 7.5 (A1), 17 (A2), 3.4 (A3), and 1.3 kJ/mol (A4) for subsites 1, 2, 3, and 4, respectively. The intrinsic rate constant k'int was estimated at 76 s-1.

Animals↗