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PubMed2004

Structural basis for broad substrate specificity of earthworm fibrinolytic enzyme component A.

Earthworm fibrinolytic enzyme component A (EFE-a) possesses an S1 pocket, which is typical for an elastase-like enzyme, but it can still hydrolyze varieties of substrates, and it exhibits wide substrate specificity. Former structure studies suggested that the four-residue insertion after Val(217) might endow EFE-a with this specificity. Based on the native crystal structure at a resolution of 2.3A, we improved the native crystal structure to 1.8A and determined its complex structure with the inhibitor Meo-Suc-Ala-Ala-Pro-Val-CMK at a resolution of 1.9A. The final structures show that: (1) EFE-a possesses multisubstrate-binding sites interacting with the substrates; (2) significant conformation adjustment takes place at two loops binding to the N-terminal of the substrates, which may enhance the interaction between the enzyme and the substrates. These characteristics make the substrate-specificity of EFE-a less dependent on the property of its S1-pocket and may endow the enzyme with the ability to hydrolyze chymotrypsin-specific substrates and even trypsin-specific substrates.

Amino Acid Sequence↗
PubMed2000

Structural basis for substrate specificity of protein-tyrosine phosphatase SHP-1.

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRPalpha, a member of the signal-regulatory proteins. We show that the variable beta5-loop-beta6 motif confers SHP-1 substrate specificity at the P-4 and further N-terminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in protein- tyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1.

Animals↗
PubMed1975

Substrate specificity of carboxypeptidase from Watermelon.

The substrate specificity of carboxypeptidase (F-II) purified from watermelon for various synthetic peptides and esters was examined kinetically. The enzyme showed a broad substrate specificity against various carbobenzoxy- and benzyl-dipeptides. Peptides containing glycine or proline were hydrolyzed slowly by the enzyme. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal resulted in an increase in the rate of hydrolysis. Inhibition studies with diisopropyl flurophosphate and diastereomers of carbobenzoxy-Phe-Ala demonstrated that the peptidase and esterase activities of the enzyme are both catalyzed by the same site of the enzyme molecule, but the binding sites for peptides and esters seem not to be the same. The enzyme also had amidase activity, which was optimal at pH 7.0.

Amidohydrolases↗
PubMed2000

Substrate specificity of cucumisin on synthetic peptides.

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4, n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Ala-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the increase of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentially same with the increase of n = 2 to 3. Then, it was concluded that cucumisin has a S5-S3' subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1' positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.

Amino Acid Sequence↗
PubMed1999

Substrate specificity of human glycinamide ribonucleotide transformylase.

The nucleotide substrate specificity of human glycinamide ribonucleotide transformylase, a chemotherapeutic target, has been examined. The enzyme accepts the sarcosyl analog of glycinamide ribonucleotide, carbocyclic glycinamide ribonucleotide, and two phosphonate derivatives of carbocyclic glycinamide ribonucleotide with V/K values, relative to that obtained for beta-glycinamide ribonucleotide, of 1, 27, 1.4, and 2.9%, respectively. Several other analogs of carbocyclic glycinamide ribonucleotide, namely a truncated phosphonate and 2',3'-dideoxy- and 2',3'-dideoxy-2',3'-didehydro-carbocyclic glycinamide ribonucleotide, were inhibitors of the enzyme, competitive against glycinamide ribonucleotide, with Ki values approximately 100 times higher than the Km for -glycinamide ribonucleotide. Although the results of the present study parallel those obtained previously with the avian enzyme (V. D. Antle, D. Liu, B. R. McKellar, C. A. Caperelli, M. Hua, and R. Vince (1996) J. Biol. Chem. 271, 6045-6049), quantitative differences between the two enzyme species have been uncovered.

Animals↗
PubMed1991

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.

Animals↗
PubMed2005

Substrate specificity of human hepatic udp-glucuronosyltransferases.

Five human hepatic UDP-glucuronosyltransferases (UGTs) catalyze the facilitated excretion of more than 90% of drugs eliminated by glucuronidation. The substrate specificity of these UGTs has been examined using cloned expressed enzymes and liquid chromatography-mass spectrometry assays to determine the intrinsic clearance of drug glucuronidation in vitro. Specific substrates for the five individual UGTs have been identified. These five probe substrates could be used to predict the drug clearance catalyzed by individual UGTs in vivo.

Gas Chromatography-Mass Spectrometry↗
PubMed2004

Kinetic analysis of precursor M1 RNA molecules for exploring substrate specificity of the N-terminal catalytic half of RNase E.

To gain insight into the mechanism by which the sequence at the rne-dependent site of substrate RNA affects the substrate specificity of Escherichia coli RNase E, we performed kinetic analysis of the cleavage of precursor M1 RNA molecules containing various sequences at the rne-dependent site by the N-terminal catalytic half of RNase E (NTH-RNase E). NTH-RNase E displayed higher K(m) and k(cat) values for more specific substrates. The retention of single strandedness at the rne-dependent site was essential for cleavage efficiency. Moreover, the loss of single-strandedness was accompanied by a decrease in both the K(m) and k(cat) values.

Endoribonucleases↗
PubMed1996

The substrate specificity of tocopherol cyclase.

The substrate specificity of the enzyme tocopherol cyclase from the blue-green algae Anabaena variabilis (Cyanobacteria) was investigated with 11 substrate analogues revealing the significance of three major recognition sites: (i) the OH group at C(1) of the hydroquinone, (ii) the (E) configuration of the double bond, and (iii) the length of the lipophilic side chain. Experiments with two affinity matrices suggest that substrates approach the enzyme's active site with the hydrophobic tail.

Chemical Phenomena↗
PubMed1986

Hydrolysis of lipid monolayers and the substrate specificity of hepatic lipase.

The substrate specificities of the phospholipase and triglyceridase activities of purified rat liver hepatic lipase were compared using lipid monolayers so that the substrates were presented to the enzyme in a controlled physical state. The rate of hydrolysis of 14C-labeled lipid at constant surface pressure in the presence of hepatic lipase and fatty acid-free bovine serum albumin at 33 degrees C was determined by monitoring the decrease of surface radioactivity. In monolayers of sphingomyelin/cholesterol (2:1, mol/mol) containing either 1 mol% triacylglycerol, 1 mol% phosphatidylethanolamine, or 10 and 20 mol% phosphatidylcholine, hepatic lipase clearly showed a preference for unsaturated over saturated lipids. In addition, with a sphingomyelin/cholesterol (2:1) monolayer containing 1 mol% of lipid substrate, hepatic lipase showed the following preference: triolein = dioleoylphosphatidylethanolamine much greater than dioleoylphosphatidylcholine; the respective rates of hydrolysis were 15.3 +/- 1.2, 14.9 +/- 0.8, and 0.5 +/- 0.1 mumol fatty acid produced/h per mg hepatic lipase. Overall, it appears that when comparing rates of hydrolysis of molecules within a given lipid class, hydrocarbon chain interactions are important. However, when comparing different lipid classes such as phosphatidylcholines and phosphatidylethanolamines, it is apparent that the polar group has a significant influence on the rate of hydrolysis. The rate of [14C]triolein hydrolysis, when mixed at surface concentrations of up to 2 mol% in a sphingomyelin/cholesterol (2:1) monolayer, was significantly faster than when triolein was present in a 1-oleyl-2-palmitylphosphatidylcholine monolayer; the rates of hydrolysis were 47.7 +/- 5.4 and 8.9 +/- 0.8 mumol fatty acid produced/h per mg hepatic lipase, respectively. The monolayer physical state and the miscibility of the substrate in the inert matrix influence the presentation of the substrate to the enzyme, thereby affecting the hydrolysis rate.

Animals↗
PubMed1996

The roles of individual amino acids in altering substrate specificity of the P450 2a4/2a5 enzymes.

A single amino acid substitution is sufficient to alter substrate specificity of P450 enzymes. Mouse P450 2a5, for example, has its substrate specificity converted from coumarin 7- to testosterone 15 alpha-hydroxylase activity by the substitution of Phe at position 209 to Leu. Furthermore, placing Asn at this position confers a novel corticosterone 15 alpha-hydroxylase activity to this P450. Recent site-directed mutational studies show the presence of the topologically common residues, each of which can determine the specificities of various mammalian P450s. For instance, residue 209 (in 2a5) corresponds to a residue at position 206 in rat P4502B1 that regulates its steroid hydroxylase activity. High substrate specificity often observed in an individual P450, therefore, can be determined and altered by the identities of a few critical residues. The structural flexibility of the substrate-heme pocket may also provide P450 enzymes with the ability to display a broad range of substrate specificities. Understanding the underlying principles whereby the flexible pocket determines P450 activities may lead us to the prediction of P450 activities based on the identities of key amino acid residues.

Amino Acids↗
PubMed1992

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.

Aldehyde Dehydrogenase↗
PubMed1993

Substrate specificity of the protein tyrosine phosphatases.

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR988-998, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR988-998 (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affinity binding and catalysis. The Km value for EGFR988-998 decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.

Amino Acid Sequence↗
PubMed1991

Dissecting the molecular mechanism of ion-solute cotransport: substrate specificity mutations in the putP gene affect the kinetics of proline transport.

Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of the putP gene, suggesting that they may replace amino acids at the active site of the enzyme. If putP substrate specificity mutations directly after the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of three putP substrate specificity mutants. One class of mutation increases the Km over 120 fold but only decreases the Vmax fourfold. Such Km mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases the Vmax 80-fold without changing the Km. Vmax mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both the Km and Vmax of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport.

Amino Acid Transport Systems, Neutral↗
PubMed1999

On the substrate specificity of DNA methyltransferases. adenine-N6 DNA methyltransferases also modify cytosine residues at position N4.

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.

Adenine↗
PubMed1989

[A membrane-bound alanine aminopeptidase from Acinetobacter calcoaceticus. 2. Substrate specificity of the enzyme].

The substrate specificity of the membrane-bound alanine aminopeptidase from Acinetobacter calcoaceticus was investigated with a series of substituted amides of alpha-amino acids. In contrast to mammalian AAP forms, the rate of hydrolysis of the AAP from Acinetobacter calcoaceticus is higher using 4-nitranilide than 2-naphthylamide substrates. The enzyme affinity is very high for alanine substrates and decreases in the series of the amide substituents, from methyl coumarylamides, 4-nitranilides, 4-methoxynaphthylamides to the 2-naphthylamides. The alanine aminopeptidase shows its highest affinity to Ala-MCA (Km = 77 mumol(s)/1).

Acinetobacter↗
PubMed1995

C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease.

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.

Amino Acid Sequence↗
PubMed2004

The structure of MBL-associated serine protease-2 reveals that identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.

A family of serine proteases mediates the proteolytic cascades of several defense mechanisms in vertebrates, such as the complement system, blood coagulation and fibrinolysis. These proteases usually form large complexes with other glycoproteins. Their common features are their modular structures and restricted substrate specificities. The lectin pathway of complement, where mannose-binding lectin (MBL) recognizes the carbohydrate structures on pathogens, is activated by mannose-binding lectin-associated serine protease-2 (MASP-2). We present the 2.25A resolution structure of the catalytic fragment of MASP-2 encompassing the second complement control protein module (CCP2) and the serine protease (SP) domain. The CCP2 module stabilizes the structure of the SP domain as demonstrated by differential scanning calorimetry measurements. The asymmetric unit contains two molecules with different CCP-SP domain orientations, reflecting increased modular flexibility at the CCP2/SP joint. This flexibility may partly explain the ability of the MASP-2 dimer to perform all of its functions alone, whereas the same functions are mediated by the much larger C1r2-C1s2 tetramer in the C1 complex of the classical pathway. The main scaffold of the MASP-2 SP domain is chymotrypsin-like. Eight surface loops determine the S1 and other subsite specificities. Surprisingly, some surface loops of MASP-2, e.g. loop 1 and loop 2, which form the S1 pocket are similar to those of trypsin, and show significant differences if compared with those of C1s, indicating that the nearly identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.

Binding Sites↗