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PubMed1987

The substrate specificity of dynein from Tetrahymena cilia.

The substrate specificity of the 22S dynein ATPase from Tetrahymena cilia was investigated. The 22S dynein exhibited a high specificity for ATP in terms of both apparent Km and Vmax: naturally occurring nucleoside triphosphates other than ATP were hydrolyzed slowly with an apparent Km of 0.25-1 mM, a sharp contrast to that of ATP hydrolysis (1-4 microM). Pyrophosphate was a poor inhibitor for the dynein ATPase, indicating weak affinity. Since dynein binds ATP tightly and hydrolyzes it at a high rate, a method to determine a trace amount of ATP in the presence of other nucleoside triphosphates has been developed by taking advantage of this enzymatic characteristic of dynein. The effect of P1,P5-di(adenosine-5'-)-pentaphosphate (Ap5A) on the 22S dynein ATPase was also investigated. Ap5A acted as a weak competitive inhibitor of the ciliary 22S dynein ATPase and the nonlinearity of the double-reciprocal plot of the ATPase was confirmed in the presence of Ap5A.

Adenine Nucleotides↗
PubMed2002

Transmembrane domains confer different substrate specificities and adenosine diphosphate hydrolysis mechanisms on CD39, CD39L1, and chimeras.

Members of the ecto-nucleoside triphosphate diphosphohydrolase (eNTPDase) family exhibit distinctive substrate specificities, but how such specificities are achieved by enzymes with identical putative catalytic domains is unknown. Previously we showed that H59G substitution changes CD39 from an apyrase to an adenosine diphosphatase (ADPase) in a manner that depends on intact associations of both transmembrane domains with the membrane. Here we show that the extracellular domain of CD39L1 ecto-adenosine triphosphatase (ecto-ATPase) has the same 3:1 ATP:ADP hydrolysis ratio as the extracellular domain of CD39, suggesting that the transmembrane domains are required to confer the native substrate specificities on each enzyme. As in CD39, H50G substitution has little effect on the activity of the CD39L1 extracellular domain or solubilized monomers. However, H50G substitution diminishes both ATPase and ADPase activities of native CD39L1, in contrast to its selective effect on ATPase activity in CD39, suggesting that the transmembrane domains confer different ADP hydrolysis mechanisms on CD39 and CD39L1. We then show that the transmembrane domains of CD39L1 can substitute for those of CD39 in conferring native CD39 substrate specificity and regulation of H59 but that the transmembrane domains of CD39 confer neither CD39 nor CD39L1 properties on the CD39L1 extracellular domain. These results suggest that non-apyrase conserved region residues in the extracellular domain contain the information specifying CD39 native properties but have a nonspecific requirement for two transmembrane domains to manifest the information.

Adenosine Diphosphate↗
PubMed1982

Substrate specificity of bacterial glycerophospholipid:cholesterol acyltransferase.

The substrate specificity of a bacterial analogue of the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been examined with small unilamellar liposomes and Triton mixed micelles. In contrast to LCAT, the microbial enzyme is capable of using all of the naturally occurring phospholipids as acyl donors. In general reaction rate depends more on the length or degree of unsaturation of the acyl chains than on the nature of the phospholipid head group. Among a series of disaturated phosphatidylcholines in liposomes, dilauroylphosphatidylcholine is the preferred acyl donor. Like LCAT, the enzyme will catalyze acyl transfer by using other alcohols in addition to cholesterol. Of saturated straight chain primary alcohols 1-decanol is the preferred acyl acceptor. Cholesterol, however, is a far better acceptor than any non-sterol alcohol tested. Other steroids with equatorial hydroxyls at position C-3 and trans-fused A:B rings will also act as acceptors whereas those steroids with axial hydroxyls at C-3 or cis-fused rings are inhibitors of acyl transfer. The ability of steroids to act as acyl acceptors may be due to the nature of their interaction with the phospholipid acyl donor.

Acyltransferases↗
PubMed1994

The use and clinical importance of a substrate-specific electrode for rapid determination of blood lactate concentrations.

OBJECTIVE: To determine the validity and clinical importance of a newly developed amperometric, enzymatic, substrate-specific electrode for the rapid measurement of circulating lactate concentrations. DESIGN: A prospective multiexperiment study. SETTING: The critical care medicine research laboratory, intensive care unit (ICU), emergency department (ED), and general wards of a university-affiliated hospital. PATIENTS: A total of 1218 patients and control subjects were studied on one or more occasions. INTERVENTIONS: Blood lactate concentrations, descriptive data, physiological parameters, and outcome results were determined in various patient populations. MAIN OUTCOME MEASURES AND RESULTS: Experiment 1: Lactate determinations performed with the new substrate-specific electrode were compared with two laboratory reference methods. Blood samples from 80 ICU patients and 165 ED patients formed the basis of this first experiment. There was excellent agreement between the test instrument and the two reference methods as reflected by bias (with reference method 1, 0.19 mmol/L; reference method 2, 0.09 mmol/L), precision (with reference method 1, +/- 0.47 mmol/L; reference method 2, +/- 0.34 mmol/L), and correlation data (with reference method 1, r = .92; reference method 2, r = .98). Experiment 2: The new test microchemistry instrument was used to analyze blood samples from 927 patients. The mean (SE) blood lactate concentrations in the various patient populations were 1.26 (0.04) mmol/L for control subjects (n = 85), 1.52 (0.03) mmol/L for general ward patients (n = 489; P < .001 vs normal subjects), 2.34 (0.15) mmol/L for ICU patients (n = 180; P < .001 vs normal subjects and general ward patients), and 2.44 (0.15) mmol/L for ED patients (n = 173; P < .001 vs normal subjects and general ward patients). None of the normal subjects and only one (0.2%) of 489 nonhypotensive general ward patients had a blood lactate value greater than 4 mmol/L. Circulating lactate concentrations greater than 4 mmol/L were 98.2% specific in predicting the need for hospital admission in patients presenting to the ED. Furthermore, lactate concentrations greater than 4 mmol/L were 96% specific in predicting mortality in hospitalized nonhypotensive patients. Experiment 3: Blood samples from 46 hypotensive ICU and ED patients and from 353 nonhypotensive ICU and ED patients (the latter samples were derived from experiment 2) were analyzed. A statistically significant difference was noted between the mean (SE) lactate concentration in hypotensive patients in the ICU and ED (4.75 [0.75] mmol/L) when compared with nonhypotensive ICU and ED patients (2.28 [0.10] mmol/L; P < .001). Furthermore, blood lactate values greater than 4 mmol/L were 87.5% specific in predicting mortality in hypotensive patients. CONCLUSIONS: Lactate determinations performed using the new test instrument are precise and accurate. Blood lactate concentrations greater than 4 mmol/L are unusual in normal and noncritically ill hospitalized patients and warrant concern. In hospitalized (non-ICU) nonhypotensive subjects, as well as in critically ill patients, a blood lactate concentration greater than 4 mmol/L may portend a poor prognosis.

Blood Chemical Analysis↗
PubMed2007

Engineering the substrate specificity of Staphylococcus aureus Sortase A. The beta6/beta7 loop from SrtB confers NPQTN recognition to SrtA.

The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the beta6/beta7 loop in SrtA with the corresponding domain from SrtB. The chimeric beta6/beta7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a k(cat)/K(m)(app) of 0.0062 +/- 0.003 m(-1) s(-1). This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (k(cat)/K(m)(app)(NPQTN)/k(cat)/K(m)(app)(LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the beta6/beta7 loop is an important site for substrate recognition in sortases.

Amino Acid Sequence↗
PubMed1993

The active site substrate specificity of the cAMP-dependent protein kinase.

cAMP-dependent protein kinase substrates have been synthesized employing an unusually efficient method that allows the alcohol-bearing residue to be incorporated into the peptide after solid phase peptide synthesis. These peptide substrates have been utilized to map the active site substrate specificity of the protein kinase. Only alpha- or beta-substituted alcohol-bearing residues containing the proper absolute configuration are phosphorylated by the enzyme. However, the cAMP-dependent protein kinase will phosphorylate achiral residues. The implications of the observed protein kinase substrate specificity with respect to inhibitor design are discussed.

Alcohols↗
PubMed1996

Substrate specificity of a novel serine protease from soybean [Glycine max (L.) Merrill].

The substrate specificity of a novel serine protease isolated from soybean seeds, cultivar Keburi, was investigated using various peptide-MCAs and several neuropeptides involving single and paired basic amino acid sequences. The protease was quite specific for arginine residue at the P1 site of the active center, and it recognized paired Arg-Arg and cleaved at the linkage between Arg-Arg or after Arg-Arg in peptide and protein molecules. This is the first protease in plant tissues which resembles in substrate specificity the arginine-specific serine proteinases from porcine gastric and intestinal mucosa, recognizing paired basic amino acid sequences.

Amino Acid Sequence↗
PubMed1991

Computational method for the design of enzymes with altered substrate specificity.

A combination of enzyme kinetics and X-ray crystallographic analysis of site-specific mutants has been used to probe the determinants of substrate specificity for the enzyme alpha-lytic protease. We now present a generalized model for understanding the effects of mutagenesis on enzyme substrate specificity. This algorithm uses a library of side-chain rotamers to sample conformation space within the binding site for the enzyme-substrate complex. The free energy of each conformation is evaluated with a standard molecular mechanics force field, modified to include a solvation energy term. This rapid energy calculation based on coarse conformation sampling quite accurately predicts the relative catalytic efficiency of over 40 different alpha-lytic protease-substrate combinations. Unlike other computational approaches, with this method it is feasible to evaluate all possible mutations within the binding site. Using this algorithm, we have successfully designed a protease that is both highly active and selective for a non-natural substrate. These encouraging results indicate that it is possible to design altered enzymes solely on the basis of empirical energy calculations.

Algorithms↗
PubMed1994

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase.

The Gln residue at amino acid position 102 of Bacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured by kcat/Km) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

Amino Acid Sequence↗
PubMed2005

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity.

Cysteine replacement of Asp190, Glu192 and Ser201 residues in the cytoplasmic interdomain loop of the TetA(B) tetracycline efflux antiporter from Tn10 reduces tetracycline resistance [Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). J Biol Chem 276, 20330-20339]. It was found that these Cys substitutions altered the substrate specificity of TetA(B), increasing the relative resistance to doxycycline and minocycline over that to tetracycline by three- to sixfold. Substitutions of Asp190 and Glu192 by Ala, Asn and Gln also impaired the ability of TetA(B) to mediate tetracycline resistance while Ser201Ala and Ser201Thr substitutions did not. A Leu9Phe substitution in the first transmembrane helix of TetA(B) suppressed the Ser201Cys mutation, undoing the alterations in resistance and specificity. That the interdomain loop might contact substrate during transport, as is suggested from its role in substrate specificity, is unexpected considering that the primary sequence in the loop is not conserved among a group of otherwise homologous TetA proteins. However, in the interdomain loop of 11 of 14 homologous TetA efflux proteins, computational analysis revealed a short alpha-helix, which includes some residues affecting activity and substrate specificity. Perhaps this conserved secondary structure accounts for the role of the non-conserved interdomain loop in TetA function.

Amino Acid Substitution↗
PubMed1994

Substrate specificity and subsite affinities of rabbit liver acid alpha-glucosidase.

The substrate specificity of rabbit liver acid alpha-glucosidase was investigated. The enzyme showed a wide specificity for various substrates, and hydrolyzed alpha-glucans such as glycogen and soluble starch. The k0 values (s-1) for maltose, kojibiose, nigerose, isomaltose, phenyl alpha-glucoside, panose, phenyl alpha-maltoside, soluble starch, beta-limit dextrin, amylopectin, shellfish glycogen, and rabbit liver glycogen were estimated to be 94.8, 18.8, 143, 3.6, 11.8, 27.8, 115, 99.2, 155, 83.5, 126, and 108, and the Km values (concentration of non-reducing terminal) for these substrates were 2.1, 1.8, 7.5, 36, 5.4, 1.9, 1.2, 0.90, 9.1, 1.0, 16, and 13 mM, respectively. Isomaltose and phenyl alpha-glucoside were unfavorable as substrates. The acid alpha-glucosidase is characterized by a relatively high activity toward glycogen. The k0 values (s-1) for maltotriose, -tetraose, -pentaose, -hexaose, -heptaose, and -octaose, and maltodextrin (n = 17) were 140, 140, 131, 132, 134, 132, and 74.3, and the Km values, 2.1, 1.8, 1.9, 3.4, 5.0, 4.9, 4.9 and 2.6 mM, respectively. Based on the rate parameters for the series of maltooligosaccharides, the subsite affinities (Ais) in the active site were evaluated as 0.54 (A1), 5.34 (A2), and 0.34 (A3) kcal/mol for subsites 1, 2, and 3, respectively. These three subsites were considered to be predominantly responsible for the binding of substrates to the active site.

Animals↗
PubMed1997

A new peptide conjugate as a highly specific substrate for MAP kinase.

A synthetic peptide (APRTPGGRC) cross-linked to poly-L-lysine through a carboxy-terminal cysteinyl residue was found to be a highly specific substrate for mitogen-activated protein (MAP) kinases. This peptide conjugate exhibited a much lower Km value (74 microM) than the free peptide substrate (APRTPGGRR, Km > 1 mM) previously used as a specific substrate for MAP kinases. Unlike myelin basic protein, which has been often used as a substrate for MAP kinases, this conjugate did not serve as substrate for cAMP-dependent protein kinase, protein kinase C, or multifunctional calmodulin-dependent protein kinases. Using the peptide conjugate as a substrate, MAP kinase activities in crude cell extracts were directly determined by in vitro assay and specifically detected by in-gel assay.

Animals↗
PubMed2004

The v-Src and c-Src tyrosine kinases immunoprecipitated from Rous sarcoma virus-transformed cells display different peptide substrate specificities.

In the cells transformed by Rous sarcoma virus (RSV), two Src proteins are expressed: the ubiquitous tyrosine kinase c-Src and the v-Src, the product of the transforming gene of the virus. Using three synthetic peptide substrates widely used for testing Src kinase activity, we show that they are phosphorylated with different efficiencies by the v-Src and c-Src tyrosine kinases immunoprecipitated from the tumor cell line H19. The v-Src displays higher efficiency (Vmax/Km ratio) toward all three peptides used, but the Vmax of v-Src is much lower than Vmax of c-Src with two peptides out of three. This difference in substrate specificity, if ignored, may cause misestimation of the amounts of active c-Src and v-Src in RSV-transformed cells. On the other hand, the different peptide substrate specificities may also reflect different protein substrate specificities of the v-Src and c-Src kinases in vivo.

Animals↗
PubMed2001

Structural basis for substrate specificities of cellular deoxyribonucleoside kinases.

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides and activate a number of medically important nucleoside analogs. Here we report the structure of the Drosophila deoxyribonucleoside kinase with deoxycytidine bound at the nucleoside binding site and that of the human deoxyguanosine kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila kinase has a wider substrate cleft, which may be responsible for the broad substrate specificity of this enzyme. The human deoxyguanosine kinase is highly specific for purine substrates; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases.

Adenosine Triphosphate↗
PubMed2004

Engineering a substrate-specific cold-adapted subtilisin.

One region predicted to be highly flexible for a psychrophilic enzyme, TA39 subtilisin (S39), was transferred in silico to the mesophilic subtilisin, savinase (EC 3.4.21.62), from Bacillus lentus (clausii). The engineered hybrid and savinase were initially investigated by molecular dynamic simulations at 300 K to show binding region and global flexibility. The predicted S39 region consists of 12 residues, which due to homology between the subtilisins, results in a total change of eight residues. By site-directed modifications, the region was transferred to the binding region of savinase, thus a savinase-S39 hybrid, named H5, was constructed. The designed hybrid showed the same temperature optimum and pH profile as savinase, but H5 had higher specific activity on the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) at all temperatures measured and, at the same time, H5 showed a decrease in thermostability. The H5 hybrid showed broader substrate specificity, measured at room temperature, due to an increase in catalytic efficiency on AAPF, AAPA and FAAF compared with savinase (N-succinyl-XXXX-pNA; XXXX = AAPF, AAPA and FAAF). The H5 hybrid showed increased activity at low temperature, increased binding region and global flexibility, as investigated by molecular dynamic simulations, and global destabilization from differential scanning calorimetry measurements. These psychrophilic characteristics indicated an increase in binding site flexibility, probably due to the modifications P129S, S130G, P131E, and thus we show that it is possible to increase low temperature activity and global flexibility by engineered flexibility in the binding region.

Bacillus↗
PubMed1997

Engineering of porcine pepsin. Alteration of S1 substrate specificity of pepsin to those of fungal aspartic proteinases by site-directed mutagenesis.

The S1 substrate specificity of porcine pepsin has been altered to resemble that of fungal aspartic proteinase with preference for a basic amino acid residue in P1 by site directed mutagenesis. On the basis of primary and tertiary structures of aspartic proteinases, the active site-flap mutants of porcine pepsin were constructed, which involved the replacement of Thr-77 by Asp (T77D), the insertion of Ser between Gly-78 and Ser-79 (G78(S)S79), and the double mutation (T77D/G78(S)S79). The specificities of the mutants were determined using p-nitrophenylalanine-based substrates containing a Phe or Lys residue at the P1 position. The double mutant cleaved the Lys-Phe(4-NO2) bonds, while wild-type enzyme digested other bonds. In addition, the pH dependence of hydrolysis of Lys-containing substrates by the double mutant indicates that the interactions between Asp-77 of the mutant and P1 Lys contribute to the transition state stabilization. The double mutant was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys6-Ile7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contributes to the S1 substrate specificity for basic amino acid residues in aspartic proteinases.

Amino Acid Sequence↗
PubMed2006

Modulation of infectivity in phage display as a tool to determine the substrate specificity of proteases.

Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.

Peptide Hydrolases↗
PubMed2002

Exploring the active site of amine:pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereoselectivity.

An active site model of the amine:pyruvate aminotransferase (APA) from Vibrio fluvialis JS17 was constructed on the basis of the relationship between substrate structure and reactivity. Due to the broad substrate specificity of the APA, various amino donors (chiral and achiral amine, amino acid, and amino acid derivative) and amino acceptors (keto acid, keto ester, aldehyde, and ketone) were used to explore the active site structure. The result suggested a two-binding site model consisting of two pockets, one large (L) and the other small (S). The difference in the size of each binding pocket and strong repulsion for a carboxylate in the S pocket were key determinants to control its substrate specificity and stereoselectivity. The L pocket showed dual recognition mode for both hydrophobic and carboxyl groups as observed in the side-chain pockets of aspartate aminotransferase and aromatic aminotransferase. Comparison of the model with those of other aminotransferases revealed that the L and S pockets corresponded to carboxylate trap and side-chain pocket, respectively. The active site model successfully explains the observed substrate specificity as well as the stereoselectivity of the APA.

Binding Sites↗