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At least 19 recordsLinked to original sources
PubMed1999

Cytochrome P450 substrate specificities, substrate structural templates and enzyme active site geometries.

The structural characteristics of human cytochrome P450 substrates are outlined in the light of extensive studies on P450 substrate specificity. Templates of superimposed substrates for individual P450 isozymes are shown to fit the corresponding enzyme active sites, where contacts with specific amino acid residues appear to be involved in the interaction with each structural template. Procedures leading to the evaluation of likely P450 specificity, binding affinity and rate of metabolism are described in the context of key examples in which molecular modelling appears to rationalize experimentally observed findings.

Animals↗
PubMed2004

Crystal structure of the toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1. Insight into the substrate specificity, substrate channeling, and active site tuning of multicomponent monooxygenases.

The four-component toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is capable of oxidizing arenes, alkenes, and haloalkanes at a carboxylate-bridged diiron center similar to that of soluble methane monooxygenase (sMMO). The remarkable variety of substrates accommodated by ToMO invites applications ranging from bioremediation to the regio- and enantiospecific oxidation of hydrocarbons on an industrial scale. We report here the crystal structures of the ToMO hydroxylase (ToMOH), azido ToMOH, and ToMOH containing the product analogue 4-bromophenol to 2.3 A or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the alpha2beta2gamma2 tertiary structure are notably different. Of particular interest is a 6-10 A-wide channel of approximately 35 A in length extending from the active site to the protein surface. The presence of three bromophenol molecules in this space confirms this route as a pathway for substrate entrance and product egress. An analysis of the ToMOH active site cavity offers insights into the different substrate specificities of multicomponent monooxygenases and explains the behavior of mutant forms of homologous enzymes described in the literature.

Azides↗
PubMed1999

Comparison of the substrate specificities of human liver cytochrome P450s 2C9 and 2C18: application to the design of a specific substrate of CYP 2C18.

A series of 2-aroylthiophenes derived from tienilic acid by replacement of its OCH2COOH substituent with groups bearing various functions have been synthesized and studied as possible substrates of recombinant human liver cytochrome P450s 2C9 and 2C18 expressed in yeast. Whereas only compounds bearing a negative charge acted as substrates of CYP 2C9 and were hydroxylated at position 5 of their thiophene ring at a significant rate, many neutral 2-aroylthiophenes were 5-hydroxylated by CYP 2C18 with kcat values of >2 min-1. Among the various compounds that were studied, those bearing an alcohol function were the best CYP 2C18 substrates. One of them, compound 3, which bears a terminal O(CH2)3OH function, appeared to be a particularly good substrate of CYP 2C18. It was regioselectively hydroxylated by CYP 2C18 at position 5 of its thiophene ring with a KM value of 9 +/- 1 microM and a kcat value of 125 +/- 25 min-1, which are the highest described so far for a CYP 2C. A comparison of the oxidations of 3, by yeast-expressed CYP 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5, showed that only CYP 2C8, 2C18, and 2C19 were able to catalyze the 5-hydroxylation of 3. However, the catalytic efficiency of CYP 2C18 for that reaction was considerably higher (kcat/KM value being 3-4 orders of magnitude larger than those found for CYP 2C8 and 2C19). Several human P450s exhibited small activities for the oxidative O-dealkylation of 3. The four recombinant CYP 2Cs were the best catalysts for that reaction (kcat between 1 and 5 min-1) when compared to all the P450s that were tested, even though it is a minor reaction in the case of CYP 2C18. All these results show that compound 3 is a new, selective, and highly efficient substrate for CYP 2C18 that should be useful for the study of this P450 in various organs and tissues. They also suggest some key differences between the active sites of CYP 2C9 and CYP 2C18 for substrate recognition.

Aryl Hydrocarbon Hydroxylases↗
PubMed2004

Partial purification and characterization of a non-cyanobacterial cyanophycin synthetase from Acinetobacter calcoaceticus strain ADP1 with regard to substrate specificity, substrate affinity and binding to cyanophycin.

This study reports, for the first time, purification and biochemical characterization of a cyanophycin synthetase from a non-cyanobacterial strain. Cyanophycin synthetase of Acinetobacter calcoaceticus strain ADP1 was purified 69-fold from recombinant Escherichia coli by two chromatographic steps and one novel affinity step utilizing the Mg(2+)-dependent binding of the enzyme to cyanophycin. Unlike cyanobacterial cyanophycin synthetases characterized so far, the purified enzyme from A. calcoaceticus strain ADP1 did not accept lysine as an alternative substrate to arginine. The apparent K(m)-values for arginine (47 microM) and aspartic acid (240 microM) were similar to those of known cyanophycin synthetases from cyanobacteria, but this enzyme had a slightly higher affinity for aspartic acid. In addition, the two different ATP-binding sites of the enzyme were characterized independently of each other with respect to K(m) values for ATP. The ATP-binding site responsible for the addition of arginine was found to have a much higher affinity for ATP (38 microM) than that responsible for the addition of aspartate (210 mM). Furthermore, the binding of the enzyme to the two possible forms of cyanophycin granule polypeptide (CGP), CGP-Asp and CGP-Arg, was studied. While both forms bound around 30-40 % of the enzyme activity present under the assay conditions, binding was Mg(2+)-dependent in the case of CGP-Asp. Two-dimensional gel electrophoresis revealed that both forms of cyanophycin were equally abundant in cyanophycin-accumulating cells of A. calcoaceticus ADP1.

Acinetobacter calcoaceticus↗
PubMed1975

Penicillinamidohydrolase in Escherichia coli. I. Substrate specificity.

Substrate specificity of the bacterial penicillinamidohydrolase (penicillinacylase, EC 3.5.1.11) from Escherichia coli was determined by measuring initial rates of enzyme hydrolysis of different substrates within zero order kinetics. Some N-phenylacetyl derivatives of amino acids and amides of phenylacetic acid and phenoxyacetic acid of different substituted amides of these acids or amides, structurally and chemically similar to these compounds, served as substrates. Significant differences in ratios of initial rates of the enzyme hydrolysis of different substrates were found using a toluenized suspension of bacterial cells or a crude enzyme preparation, in spite of the fact that the enzyme is localized between the cell wall and cytoplasmic membrane, in the so-called periplasmic space. N-phenylacetyl derivatives are the most rapidly hydrolyzed substrates. Beta-phenylpropionamide and 4-phenylbutyramide were not utilized as substrates. The substrate specificity of the enzyme is discussed with respect to a possible use of certain colourless compounds as substrates, hydrolysis of which yields chromophor products suitable for a simple and rapid assay of the enzyme activity.

Amides↗
PubMed1993

Plant mitochondrial RNase P and E. coli RNase P have different substrate specificities.

Substrate specificity requirements of the plant mitochondrial RNase P were investigated with different natural and mutated substrates. Heterologous precursors with intact tRNAs from plant and fungal mitochondria, from bacteria, archaebacteria and of eukaryotic origins were processed faithfully, albeit with different efficiencies. Alteration of the acceptor stem length did not disturb correct processing, while activity is completely inhibited by deletion of the pseudouridine loop. Such and other minimal substrates processed by the E. coli RNase P are not recognized as substrates by the plant mitochondrial enzyme, suggesting different requirements for substrate recognition.

Culture Techniques↗
PubMed1991

8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity.

Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodG.dA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG.dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.

Base Sequence↗
PubMed1994

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.

Amino Acid Sequence↗
PubMed2002

Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates.

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs.

Acyltransferases↗
PubMed1984

Localization in mammalian brain of G-substrate, a specific substrate for guanosine 3',5'-cyclic monophosphate-dependent protein kinase.

The regional and cellular distribution of G-substrate, a 23,000-dalton protein substrate specific for guanosine 3',5'-cyclic monophosphate-dependent protein kinase, has been examined in mammalian brain using immunoprecipitation, radioimmunoassay, and peptide-mapping techniques. In rabbit brain, G-substrate was found to be highly concentrated in the cerebellum. The concentration of G-substrate in cerebellar cytosol was 27.2 pmol/mg. The concentrations of G-substrate in cortex, hippocampus, and caudate were only 1 to 2% of that found in cerebellum. Studies of neurological mutant mice lacking either Purkinje cells (PCD and nervous) or granule cells (weaver) suggested that, within the cerebellum, G-substrate is localized almost exclusively in Purkinje cells. A phosphoprotein present in noncerebellar brain regions, which co-migrated with G-substrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown by peptide mapping to consist predominantly of phosphatase inhibitor-1. Phosphatase inhibitor-1, a potent inhibitor of protein phosphatase-1, is known to share several physicochemical properties with G-substrate. In contrast to the results obtained with G-substrate, the concentration of phosphatase inhibitor-1 was significantly lower in cerebellum than in other major brain regions. These and other data suggest that G-substrate may be a Purkinje cell-specific protein phosphatase inhibitor.

Amino Acids↗
PubMed2001

Beta D305A mutant of tryptophan synthase shows strongly perturbed allosteric regulation and substrate specificity.

Substrate channeling in the tryptophan synthase bienzyme is regulated by allosteric interactions. Allosteric signals are transmitted via a scaffolding of structural elements that includes a monovalent cation-binding site and salt-bridging interactions between the side chains of betaAsp 305, betaArg 141, betaLys 167, and alphaAsp 56 that appear to modulate the interconversion between open and closed conformations. betaAsp 305 also interacts with the hydroxyl group of the substrate L-Ser in some structures. One possible functional role for betaAsp 305 is to ensure the allosteric transmission that triggers the switching of alphabeta-dimeric units between open and closed conformations of low and high activity. This work shows that substitution of betaAsp 305 with Ala (betaD305A) decreases the affinity of the beta-site for the substrate L-Ser, destabilizes the enzyme-bound alpha-aminoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleophile specificity of the beta-reaction. The altered specificity provides a biosynthetic route for new L-amino acids derived from substrate analogues. betaD305A also shows an increased rate of formation of pyruvate upon reaction with L-Ser relative to the wild-type enzyme. The formation of pyruvate is strongly inhibited by the binding of benzimidazole to E(A-A). Upon reaction with L-Ser and in the presence of the alpha-site substrate analogue, alpha-glycerol phosphate, the Na(+) form of betaD305A undergoes inactivation via reaction of nascent alpha-aminoacrylate with bound PLP. This work establishes important roles for betaAsp 305 both in the conformational change between open and closed states that takes place at the beta-site during the formation of the E(A-A) and in substrate binding and recognition.

Alanine↗
PubMed2002

G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition.

To understand the specific genetic instabilities associated with deficiencies in RecQ family helicases, we have studied the substrate preferences of two closely related members of this family, human BLM and Saccharomyces cerevisiae Sgs1p. Here we show that both BLM and Sgs1p preferentially unwind G4 DNA relative to Holliday junction substrates, and that substrate preference reflects binding affinity and maps to the conserved central helicase domain. We identify the porphyrin N-methyl mesoporphyrin IX (NMM) as a specific inhibitor of G4 DNA unwinding, and show that in the presence of NMM the helicase becomes trapped on the NMM-G4 DNA complex, consuming ATP but unable to unwind or dissociate. These results suggest that BLM and Sgs1p function proactively in replication to remove G4 DNA structures which would otherwise present obstacles to fork progression, rather than by promoting recombination to restart a fork that has stalled.

Adenosine Triphosphatases↗
PubMed2000

Theoretical investigation of substrate specificity for cytochromes P450 IA2, P450 IID6 and P450 IIIA4.

Three-dimensional models of the cytochromes P450 IA2, P450 IID6 and P450 IIIA4 were built by means of comparative modeling using the X-ray crystallographic structures of P450 CAM, P450 BM-3, P450 TERP and P450 ERYF as templates. The three cytochromes were analyzed both in their intrinsic structural features and in their interaction properties with fifty specific and non-specific substrates. Substrate/enzyme complexes were obtained by means of both automated rigid and flexible body docking. The comparative analysis of the three cytochromes and the selected substrates, in their free and bound forms, allowed for the building of semi-quantitative models of substrate specificity based on both molecular and intermolecular interaction descriptors. The results of this study provide new insights into the molecular determinants of substrate specificity for the three different eukaryotic P450 isozymes and constitute a useful tool for predicting the specificity of new compounds.

Amino Acid Sequence↗
PubMed1985

Specific substrate for histone kinase II: a synthetic nonapeptide.

Based on the previously determined intrinsic substrate specificity of histone kinase II, a nonapeptide was synthesized which was a specific substrate for this enzyme. The Vmax value of phosphorylation of the peptide (Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide) was about the same as that for H1 histone and the apparent Km for the phosphorylation of the peptide was 0.2 mM, an order of magnitude higher than that for H1 histone. H1 histone inhibited the phosphorylation of the peptide, while the peptide did not inhibit the phosphorylation of H1 histone. In the crude extracts of calf thymus, spleen and liver, histone kinase II was the only enzyme which phosphorylated the synthetic peptide. The rate of phosphorylation of this peptide was used to determine the activity of histone kinase II in the crude extracts of several tissues obtained from different species.

Animals↗
PubMed1994

The structure of the transglutaminase 1 enzyme. Deletion cloning reveals domains that regulate its specific activity and substrate specificity.

Transglutaminase 1 (TGase1) is one of three known enzymes involved in terminal differentiation in stratified squamous epithelia, possibly in the formation of a cornified cell envelope. Because the intact enzyme is particularly difficult to isolate in quantity from keratinocytes for characterization, comparatively little is known about its properties. We have expressed the full-length as well as a series of deletion forms of this enzyme in a bacterial system and analyzed their enzymatic properties. The specific activity of the full-length enzyme isolated and purified from the bacterial lysate was comparable to that of the native enzyme of keratinocytes. Analysis of several deletion constructs demonstrated that removal of the first 60-109 residues, which include sequences involved in membrane association, results in upwards of a 10-fold increase in the specific activity. Deletions beyond residue 109, into sequences conserved within the TGase family of proteins, result in loss of activity. Similarly, as many as 240 residues can be removed from its carboxyl-terminal end before activity is lost. Thus, a molecule of 466 residues, containing virtually only the conserved core sequences of TGases, retains a specific activity comparable to the intact enzyme. In addition, the various deletion forms display wide variations in substrate specificity toward a series of synthetic peptide substrates, designed from possible target TGase1 substrate proteins of epithelia. The data show that sequences between residues 62 and 92 are important in defining the substrate specificity of the TGase1 enzyme system. Furthermore, it may now be possible to design an enzyme with a defined substrate specificity. Together, these data suggest TGase1 has recruited additional sequences on its amino terminus in relation to other members of the TGase family, which have the net effect of permitting sequestration onto membranes, changing its specific activity and modifying its likely substrate specificities.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of the hemagglutinin receptor specificity and neuraminidase substrate specificity of clinical isolates of human influenza A viruses.

Six clinical isolates of influenza A viruses were examined for hemagglutinin receptor specificity and neuraminidase substrate specificity. All of the viral isolates minimally passaged in mammalian cells demonstrated preferential agglutination of human erythrocytes enzymatically modified to contain NeuAc alpha 2,6Gal sequences, with no agglutination of cells bearing NeuAc alpha 2,3Gal sequences. This finding is consistent with the hemagglutination receptor specificity previously demonstrated for laboratory strains of influenza A viruses. The neuraminidase substrate specificities of the clinical isolates examined were also identical to that described for the N2 neuraminidase of recent laboratory strains of human influenza viruses. The H3N2 viruses all displayed the ability to release sialic acid from both alpha 2, 3 and alpha 2, 6 linkages. In addition, two clinical isolates of H1N1 viruses also demonstrated this dual neuraminidase substrate specificity, a characteristic which has not been previously described for the N1 neuraminidase. These results demonstrate that complementary hemagglutinin and neuraminidase specificities are found in recent isolates of both H1N1 and H3N2 influenza viruses.

HN Protein↗
PubMed2001

Stereo-specific substrate recognition by phosphatidylinositol phosphate kinases is swapped by changing a single amino acid residue.

Type I and type II phosphatidylinositol phosphate (PIP) kinases generate the lipid second messenger phosphatidylinositol (PtdIns) 4,5-bisphosphate and thus play fundamental roles in the regulation of many cellular processes. Although the two kinase families are highly homologous, they phosphorylate distinct substrates and are functionally non-redundant. Type I PIP kinases phosphorylate PtdIns 4-phosphate at the D-5 hydroxyl group and are consequently PtdIns 4-phosphate 5-kinases. By contrast, type II PIP kinases are PtdIns 5-phosphate 4-kinases that phosphorylate PtdIns 5-phosphate at the D-4 position. Type I PIP kinases, in addition, also phosphorylate other phosphoinositides in vitro and in vivo and thus have the potential to generate multiple lipid second messengers. To understand how these enzymes differentiate between stereoisomeric substrates, we used a site-directed mutagenesis approach. We show that a single amino acid substitution in the activation loop, A381E in IIbeta and the corresponding mutation E362A in Ibeta, is sufficient to swap substrate specificity between these PIP kinases. In addition to its role in substrate specificity, the type I activation loop is also key in subcellular targeting. The Ibeta(E362A) mutant and other mutants with reduced PtdIns 4-phosphate binding affinity were largely cytosolic when expressed in mammalian cells in contrast to wild-type Ibeta which targets to the plasma membrane. These results clearly establish the role of the activation loop in determining both signaling specificity and plasma membrane targeting of type I PIP kinases.

Amino Acid Sequence↗