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PubMed2005

Substrate specificity of protein kinases and computational prediction of substrates.

To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of "peptide specificity" of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information.

Binding Sites↗
PubMed1982

Intraspecies and interspecies variations in the substrate specificity of D-amino acid oxidase.

1. Substrate specificity of D-amino acid oxidase was examined in the kidney homogenates of the mouse and the six species of fishes. 2. The enzyme of the mouse did not show a significant intraspecies variation in the substrate specificity. The sex and age of the mouse did not affect the substrate specificity of the enzyme. 3. The degree of similarity in the substrate specificity of the enzymes was quantified as the variation index. The values of the variation index among the enzymes of the fishes paralleled their taxonomic relatedness.

Animals↗
PubMed1995

Trichodiene synthase. Substrate specificity and inhibition.

The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70.

Binding, Competitive↗
PubMed2003

Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity.

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.

Amino Acid Sequence↗
PubMed2001

Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity.

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis. When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type. The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.

Amino Acid Sequence↗
PubMed1986

[Comparative characteristics of soluble and membrane brain aminopeptidases. II. Substrate specificity].

Comparative studies on substrate specificity of the soluble and membrane-bound aminopeptidases from bovine brain were carried out. A series of p-nitroanilides and beta-naphthylamides of amino acids, di- and tripeptides with the aminoterminal phenylalanine residue, as well as a biologically active pentapeptide--[Leu5]enkephalin--were used as substrates. The soluble and membrane-bound aminopeptidases manifested identical specificity towards the employed substrates. The aminopeptidases were equally effective towards the p-nitroanilides of amino acids and peptides, whereas beta-naphthylamides were more susceptible to hydrolysis by both aminopeptidases than p-nitroanilides and peptides. Taking into account physico-chemical characteristics of these enzymes, it was concluded that the soluble and membrane-bound aminopeptidases are quite similar or perhaps identical. Their role in the regulation of nervous system functioning was discussed. A comparison of specificities for brain aminopeptidases and leucine aminopeptidase from bovine lens led to the conclusion that they belong to different groups. This feature allows planning the synthesis of selective inhibitors.

Amino Acids↗
PubMed1987

Biliverdin reductase: substrate specificity and kinetics.

The substrate specificity of the different forms of rat liver biliverdin reductase was examined using synthetic biliverdins. Biliverdins carrying methyl, ethyl and one propionate residue in their structure were not substrates of biliverdin reductase. Biliverdins with one propionate and one acetate residue or with two acetate residues were not reduced by the enzyme either. The presence of two propionates in the biliverdin structure gave a biliverdin with substrate activity. Increasing the number of propionates to four, as in coprobiliverdins, did not affect substrate activity, while the octaacid urobiliverdins were also good substrates of the enzymes. The beta isomer of urobiliverdin III and coprobiliverdin III were reduced at much higher rates by molecular form 3 of the enzyme as compared to molecular form 1, a fact which had already been observed with the beta isomer of biliverdins IX, XIII and hematobiliverdin. All the biliverdins mentioned above were readily reduced to bilirubins by sodium borohydride. The purified molecular forms 1 and 3 displayed sigmoidal kinetics with most of the biliverdins tested. The data were analyzed by nonlinear regression in a microcomputer and it was found that they fitted a model of a moderate cooperative dimer where both ES and ES2 are catalytically active. The Vm, Ks and the Hill numbers, nH, for biliverdin IX alpha and beta, hematobiliverdin IX alpha and beta, and several synthetic biliverdin isomers are given. Molecular form 2 showed classical Michaelian kinetics.

Algorithms↗
PubMed2006

LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity.

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

Amino Acid Sequence↗
PubMedDate not supplied

Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity.

To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.

Amino Acid Sequence↗
PubMed1984

[Aminoacylase from Streptoverticillium microorganisms: stereo- and substrate specificity].

The stereo- and substrate specificity of a new aminoacylase from Streptoverticillium microorganisms was studied. The enzyme effectively hydrolyzes acetyl derivatives of aliphatic (methionine, leucine) and aromatic (phenylglycine, phenylalanine, tryptophan) amino acids. The L-enanthiomer of acetylphenylglycine is hydrolyzed by aminoacylase 8000 times more effectively than the D-enanthiomer. A procedure for determination of the enanthioselectivity of aminoacylases was elaborated. This procedure is designed for a detection and assessment of contaminations of the N-acetyl derivative of one enanthiomer by another enanthiomer of the amino acid, as well as of the degree of racemization of the substrate during hydrolysis of acetyl derivatives of D-amino acids.

Amidohydrolases↗
PubMed2002

A modular assembly strategy for improving the substrate specificity of small catalytic peptides.

In contrast to large proteins, small peptide catalysts typically display limited specificity for small molecule substrates. This is presumably a result of the limited opportunities small peptides have to fold in a manner that provides for the formation of an isolated reaction vessel that effectively binds and sequesters substrates from bulk solvent while at the same time catalyzing their transformation. For the preparation of small peptide catalysts that possess improved substrate specificity, we have developed a modular assembly strategy that involves appending phage display-derived substrate binding-domain modules to catalytically active peptide domains. We demonstrate the potential of this strategy with the construction of a small 35-amino acid residue aldolase peptide with improved substrate specificity. The advantages of this approach are that it reduces the demand on the functionalization of the catalytic site and it is modular, therefore making its adaptation to a variety of specificities rapid. The modular assembly strategy studied here may present advantages over exhaustive searches of large random-sequence peptide libraries for peptides with singular function.

Amino Acid Sequence↗
PubMed1983

Substrate specificity of S-adenosylhomocysteinase. Cysteine is a substrate of the plant and mammalian enzymes.

Substrate specificity of S-adenosylhomocysteinases (S-adenosyl-L-homocysteine hydrolase, EC 3.3.1.1) with respect to amino acid has been studied using homogeneous preparations of the enzymes from yellow lupin (Lupinus luteus) seeds and bovine liver. Both enzymes use cysteine, in addition to homocysteine, as a substrate. Homoserine, serine, pinicillamine, reduced glutathione and 2-mercaptoethanol are not substrates. In the presence of cysteine, the reaction of S-adenosylthio-amino acid synthesis is characterized by 20-40-fold lower kcat values (kcat = 0.23 s-1 or 0.11 s-1 in the presence of cysteine and either bovine or lupin enzyme) and 270-250-fold higher Km values (Km for cysteine is 15 mM and 35 mM with bovine and lupin enzyme, respectively) than the reaction in the presence of the normal substrate, homocysteine. In the reverse reaction, S-adenosylcysteine is hydrolyzed by the mammalian enzyme much faster than by the plant one. Specificity (kcat/Km) towards S-adenosylcysteine and S-adenosylhomocysteine is 0.9 M-1 . s-1 and 60 000 M-1 . s-1, respectively, with the plant enzyme and 15.3 M-1 . s-1 and 70 000 M-1 . s-1, respectively, with the mammalian enzyme. With plant enzyme, the reactions with cysteine and homocysteine are not competitive, i.e., cysteine does not inhibit the synthesis of S-adenosylhomocysteine, and homocysteine does not inhibit the synthesis of S-adenosylcysteine. This is consistent with independent binding of cysteine and homocysteine to both enzyme subunits. Using adenosine analogs and the mammalian S-adenosylhomocysteinase we were able to synthesize a number of novel S-adenosylcysteine analogs. These included: S-N6-hydroxyadenosyl-L-cysteine, S-2-aminoadenosyl-L-cysteine, S-nebularyl-L-cysteine, S-3-deazaadenosyl-L-cysteine, S-formycyl-L-cysteine, S-N6-methyladenosyl-L-cysteine and S-N1-oxideadenosyl-L-cysteine.

Adenosylhomocysteinase↗
PubMed1999

Substrate specificities and identification of putative substrates of ATM kinase family members.

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.

Amino Acid Sequence↗
PubMed1998

Substrate specificity of sheep liver sorbitol dehydrogenase.

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

Animals↗
PubMed1994

Substrate specificity of porcine renin: P1', P1, and P3 residues of renin substrates are crucial for activity.

Renin, the rate-limiting enzyme in the formation of angiotensin II, is well-known for its stringent substrate specificity. In this study, the biochemical basis for the unusual specificity of renin was investigated by replacing individual amino acids in the octapeptide substrate of renin with Ala. Kinetic analyses of Ala-substituted substrates revealed that the substitutions did not cause significant changes in the Km values, but did cause variable changes in the kcat and kcat/Km values. Ala substitutions at the P1', P1, and P3 sites decreased the kcat/Km values by 400-700-fold. Similar substitutions at the P3', P2, P4, and P5 sites only reduced the kcat/Km values by 2-7-fold. Interestingly, Ala substitution for the P2' Val produced a substrate with an approximately 3-fold increase in activity. These results indicate that the P1', P1, and P3 residues are crucial in determining the substrate specificity of renin. The findings also suggest that the specificity of renin is achieved mainly through substrate discrimination in the transition state, rather than in the ground state. Further studies on the effects of amino acid substitutions at the P2' site revealed that non-branched-chain amino acids (e.g., Ala and alpha-aminobutyric acid) are preferred at this site. Only P1' substitution demonstrated any significant change in Km, presumably due to the decreased hydrophobic interactions in the S1' site upon Ala substitution. The species specificity of renin presumably arises from differing P1'-P3' residues in angiotensinogens. For example, the P1'-P3' residues from human and porcine angiotensinogens are Ile-Val-His and Leu-Val-Tyr, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed2005

Comparison of the substrate specificity of two potyvirus proteases.

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.

Amino Acid Sequence↗
PubMed1980

The 5'-nucleotidase activity in normal human serum. Electrophoretic patterns and substrate specificity.

Both the electrophoretic patterns and substrate specificity of the total 5'-nucleotidase activity at pH 7.4 in serum from 10 normal adults (19-49 years) were identical using various purine and pyrimidine mononucleotides as substrates. Different purified alkaline phosphatases were studied at the same time in the same manner. The serum enzymes showed substrate specificity qualitatively similar to that of the bovine liver enzyme. The electrophoretic study showed two fractions for both the liver enzyme and the serum enzymes with the different substrates, indicating that each of these are dephosphorylated by the same two, mutually different, enzyme molecules. Each of the other enzymes showed only a single fraction, which was identical for each enzyme with the various substrates.

5'-Nucleotidase↗
PubMed1979

Substrate-specificity of uvr excision repair.

The substrate specificity of the uvr endonuclease, the product of the uvrA, uvrB, and uvrC genes is reviewed. It is suggested that the relatively well-defined substrate specificity of this repair enzyme is useful as a guide in determining the nature of the DNA-lesion caused by a given mutagen.

DNA Polymerase I↗