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PubMed1994

P450 in the rat and man: methods of investigation, substrate specificities and relevance to cancer.

1. Considerable evidence has been accumulated that orthologous rat and human P450 forms oxidize numerous chemicals in a highly similar manner, including the detoxication and activation of mutagens and carcinogens. 2. Nevertheless, certain specific substrates of rat P450s are not so well oxidized by the orthologous human forms, and vice versa. 3. Certain mutagens and carcinogens can be activated in a similar way by different (non-orthologous) forms in rat and man, confirming that studies on animals, directed ultimately to man, can be indicative but not predicative of chemical mutagenesis and carcinogenesis.

Animals↗
PubMed1986

Amino acid specific ADP-ribosylation: substrate specificity of an ADP-ribosylarginine hydrolase from turkey erythrocytes.

An ADP-ribosylarginine hydrolase, which catalyzes the degradation of ADP-ribosyl[14C]arginine to ADP-ribose plus arginine, was separated by ion exchange, hydrophobic, and gel permation chromatography from NAD:arginine ADP-ribosyltransferases, which are responsible for the stereospecific formation of alpha-ADP-ribosylarginine. As determined by NMR, the specific substrate for the hydrolase was alpha-ADP-ribosylarginine, the product of the transferase reaction. The ADP-ribose moiety was critical for substrate recognition; (phosphoribosyl) [14C]arginine and ribosyl[14C]arginine were poor substrates and did not significantly inhibit ADP-ribosyl[14C]arginine degradation. In contrast, ADP-ribose was a potent inhibitor of the hydrolase and significantly more active than ADP greater than AMP greater than adenosine. In addition to ADP-ribosyl[14C]arginine, both ADP-ribosyl[14C]guanidine and (2'-phospho-ADP-ribosyl)[14C]arginine were also substrates; at pH greater than 7, ADP-ribosyl[14C]guanidine was degraded more readily than the [14C]arginine derivative. Neither arginine, guanidine, nor agmatine, an arginine analogue, was an effective hydrolase inhibitor. Thus, it appears that the ADP-ribosyl moiety but not the arginine group is critical for substrate recognition. Although the hydrolase requires thiol for activity, dithiothreitol accelerated loss of activity during incubation at 37 degrees C. Stability was enhanced by Mg2+, which is also necessary for optimal enzymatic activity. The findings in this paper are consistent with the conclusion that different enzymes catalyze ADP-ribosylarginine synthesis and degradation. Furthermore, since the hydrolase and transferases possess a compatible stereospecificity and substrate specificity, it would appear that the two enzymatic activities may serve as opposing arms in an ADP-ribosylation cycle.

Adenine Nucleotides↗
PubMed1998

Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity.

The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity. Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif. Within this segment, we identified two sets of residues [ERGA --> DKGT (522-525) and T578C] that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein [Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170]. We wished to gain insight into the molecular basis of this defect. For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris. P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes. P-gp proteoliposomes from P. pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells. Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion. Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity. Photolabeling studies with [125I]iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants. The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

Determination of the specific substrate sequence motifs of protein kinase C isozymes.

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, mu, and zeta) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKC mu, selected peptides with basic amino acids at positions -6, -4, and -2. PKC alpha, -betaI, -betaII, -gamma, and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKC delta, -epsilon, -zeta, and -mu preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKC alpha, -gamma, and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC mu showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKC alpha, -betaI, -betaII, -gamma, -delta, and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC alpha, -betaI,-delta, -zeta, and -mu were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

Amino Acid Sequence↗
PubMed1994

[Reexamination of BT-PABA test (PFD)--on the substrate specificity for pancreatic chymotrypsin in vivo].

It is widely believed that BT-PABA is a specific substrate for pancreatic chymotrypsin, and based on this concept, BT-PABA test is used to evaluate the exocrine pancreatic function. But this test shows unusual clinical results particularly in patients after total pancreatectomy. In order to reevaluate the specificity of BT-PABA for chymotrypsin, we investigated the intestinal influence on this substrate in in vivo and in vitro studies using mongrel dogs under the conditions of complete absence of pancreatic juice in the alimentary tract. In vivo study showed that serum PABA increased after BT-PABA administration in the models of complete absence of pancreatic chymotrypsin and in vitro study demonstrated BT-PABA splitting activity fo jejunum and ileum mucosal homogenate which increased after total pancreatectomy. Hence, we concluded that BT-PABA is not a specific substrate for pancreatic chymotrypsin in vivo in human and in dog.

4-Aminobenzoic Acid↗
PubMed1981

A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. I. Purification and characterization.

A protein that exhibits greater substrate specificity for cGMP-dependent protein kinase than for cAMP-dependent protein kinase has been purified 8,000-fold from cytosol of rabbit cerebellum to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein, termed G-substrate, is a monomer of 23,000 daltons. It is heterogeneous on isoelectric focusing, exhibiting three isoelectric forms over the pH range of 5.2-5.6 cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of G-substrate, both into threonine residues. The protein has a high content of aspartate, glutamate, and proline. The hydrodynamic properties, heat stability, and acid solubility of this protein are consistent with an unfolded, nonglobular structure. G-substrate is localized primarily in the cytosol of cerebellum, although low concentrations of a phosphorylated protein with a similar molecular weight are detected in other brain regions.

Amino Acids↗
PubMed1997

Prediction of substrate-specific pockets in cyclosporin synthetase.

Amino acid sequence comparisons between domains of cyclosporin synthetase have been used to identify regions of the sequence which are responsible for the recognition and binding of the individual amino acids. Using a limited set of selection rules it was possible to identify three amino acid positions in the subdomain sequences which are responsible for amino acid specificity. Homology with the firefly luciferase protein shows that these three key residues are close to each other and line the surface of a putative specific substrate binding pocket located on the amino acyl-adenylation subdomain. These results allow us to predict a large number of cyclosporin synthetase mutants which could be used to synthesise alternative cyclosporin-like peptides.

Amino Acid Sequence↗
PubMed1993

Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells.

Flk2/Flt3 is a recently identified receptor tyrosine kinase expressed in brain, placenta, testis, and primitive hematopoietic cells. The mitogenic signalling potential and biochemical properties of Flk2/Flt3 have been analyzed by using a chimeric receptor composed of the extracellular domain of the human colony-stimulating factor 1 receptor and the transmembrane and cytoplasmic domains of murine Flk2/Flt3. We demonstrate that colony-stimulating factor 1 stimulation of the Flk2/Flt3 kinase in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a full proliferative response in the absence of other growth factors. In transfected interleukin 3 (IL-3)-dependent Ba/F3 lymphoid cells, activation of the chimeric receptor can abrogate IL-3 requirement and sustain long-term proliferation. We show that phospholipase C-gamma 1, Ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, Vav, Fyn, and Src are components of the Flk2/Flt3 signal transduction pathway. In addition, we demonstrate that phospholipase C-gamma 1, the p85 subunit of phosphatidylinositol 3'-kinase, Shc, Grb2, and Src family tyrosine kinases, but not Ras GTPase-activating protein, Vav, or Nck, physically associate with the Flk2/Flt3 cytoplasmic domain. Cell-type-specific differences in tyrosine phosphorylation of p85 and Shc are observed. A comparative analysis of the Flk2/Flt3 signal cascade with those of the endogenous platelet-derived growth factor and IL-3 receptors indicates that Flk2/Flt3 displays specific substrate preferences. Furthermore, tyrosine phosphorylation of p85 and Shc is similarly affected by totally different growth factors in the same cellular background.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.

Animals↗
PubMed1998

Directed evolution of an aspartate aminotransferase with new substrate specificities.

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.

Amino Acid Sequence↗
PubMed1989

Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition.

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.

Alanine↗
PubMed1993

Substrate specificities of cyclosporin synthetase and peptolide SDZ 214-103 synthetase. Comparison of the substrate specificities of the related multifunctional polypeptides.

The recently discovered multifunctional polypeptide cyclosporin synthetase is capable of synthesizing the cyclic undecapeptide cyclosporin A in a batch reaction. Substrates are the unmethylated constitutive amino acids of cyclosporin A. Exchange of one or more of these by various amino acids gives a picture of the substrate specificity of the enzyme in vitro, which is different from the known picture obtained by in vivo studies. The uncommon amino acid butenylmethylthreonine in position 1 of the cyclosporin ring can be exchanged by an unexpected large spectrum of different amino acids, showing a great flexibility of this site. Position 2, on the other hand, which shows the greatest variability in vivo, has an only slightly lower specificity in vitro. Position 3 has a very high degree of specificity; positions 4, 6, 7, 9, and 10 have marginally less. The variability of positions 5 and 11 is moderate, whereas position 8 shows only low substrate specificity in vitro. In general, most sites of SDZ 214-103 synthetase appear to be more specific than those of cyclosporin synthetase. Site 11 has nearly identical substrate specificity compared with that of cyclosporin synthetase. The D-2-hydroxy acid position (position 8) can be occupied by a large spectrum of substrates varying from D-lactic acid to D-2-hydroxyisocaproic acid. Within the limits of the present data, the addition of further functional groups to the D-2-hydroxy acid moiety are apparently not tolerated by the enzyme.

Amino Acid Sequence↗
PubMed1991

Rat liver polysome N alpha-acetyltransferase: substrate specificity.

The substrate specificity of polysome rat liver N alpha-acetyltransferase (NAT) has been examined by utilizing a series of synthetic and natural substrates that has been systematically altered with respect to N-terminal sequence and length. Families of peptides of the structure S-Y-S-G-G-L-L-L were generated by successively replacing the N-terminal serine, the penultimate tyrosine, and the antepenultimate serine with all 19 commonly occurring amino acids, which were then assessed for their reactivity with the rat liver enzyme. Only peptides with N-terminal serine, alanine, methionine, leucine, and phenylalanine were modified. Glycine, lysine, arginine, valine, isoleucine, and tryptophan in the second position are (with N-terminal serine) strongly inhibitory, and proline completely blocks modification. Third-position substitutions have less of an effect on NAT activity with glycine, aspartic acid, glutamic acid, and tryptophan being most inhibiting (with N-terminal Ser-Tyr). These observations are generally in agreement with in situ modifications although there are some significant differences particularly with respect to the amino-terminal residues. Optimal chain length was determined to be 10-11 residues with either synthetic peptides of the structure S-Y-S-(G)n-L-L-L or adrenocorticotropin (ACTH) sequences ranging from 8 to 39 residues. The ACTH peptides were generally found to be severalfold better substrates than the corresponding synthetic ones. Activity was not affected by increased chain length beyond approximately 17 residues. These data support the view that polysome-catalyzed N alpha-acetylation occurs as a cotranslational event on nascent chains of about 20-40 amino acids in length.

Acetylation↗
PubMed1978

Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity.

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.

Chymotrypsin↗
PubMed1982

Diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum. Substrate specificity.

The substrate specificity of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum for dinucleoside polyphosphates has been determined by high-performance liquid chromatography (HP-LC). Elution of a strong anion-exchange resin with a pH and ionic strength gradient of ammonium phosphate separates a series of monoadenosine and diadenosine polyphosphates. Most of the corresponding guanine nucleotides are also resolved on this HPLC system. One mole each of Ap4A and Gp4G is symmetrically hydrolyzed to 2 mol of ADP and GDP, respectively. Ap3A, Ap5A, Ap6A, and Ap4 are hydrolyzed, and in each case ADP is one of the products. Gp3G, Gp5G, Gp6G, and Gp4 are also substrates, and in each case GDP is one of the products. AMP, ADP, ATP, Ap2A, ADPR, GMP, GDP, GTP, NAD+, and NADP+ are not substrates. No hydrolysis of the cap dinucleotides m7Gp3Am and m7Gp3Cm was detected by HPLC. Diadenosine tetraphosphate pyrophosphohydrolase preparations were also assayed for adenylate kinase, nucleotide diphosphate kinase, NAD(P)+ pyrophosphohydrolase, phosphodiesterase, cyclic nucleotide phosphodiesterase, phosphatase, and ribonuclease activities. These enzymic activities were not detectable in diadenosine tetraphosphate pyrophosphohydrolase. The symmetrical hydrolysis of Ap4A and Gp4G is an unique catalytic property that distinguishes diadenosine tetraphosphate pyrophosphohydrolase from P. polycephalum from diadenosine tetraphosphate phosphohydrolases from other organisms.

Acid Anhydride Hydrolases↗
PubMed1980

N-Acetyltransferases of rat liver and blood: substrate specificities.

The substrate specificities of N-acetyltransferases (NAT) were investigated by measuring Vmax and Km values with p-aminobenzoic acid (I), p-aminobenzamide (II), p-amino-benzamidopyridine (III), 2-(p-aminobenzamido)-4,6-dimethylpyrimidine (VI), and the corresponding p-aminobenzenesulfonamides (VII, VIII and XI) using at rat liver and blood preparations. With liver NAT, II, III and VI had lower Km and higher Vmax values than did their corresponding sulfonyl analogs (VII, VIII, XI). III was extraordinarily active (Vmax 854 nmol/mg protein/h); in contrast, II gave a Vmax of 22.4. Sulfadiazine (IX) and sulfamerazine (X) were acetylated at a very slow rate. The activities of the blood enzymes on these compounds were very different. The Vmax values obtained with blood NAT for II, VI and VII were sharply decreased. Surprisingly, acetylation of III, VIII, IX and X could not be detected. In contrast to liver, the blood NAT gave lower values of both Vmax and Km for the S analog of II and a much higher Km for I. While p-aminobenzoic acid was the best substrate for blood NAT, substitution of the amido nitrogen of p-aminobenzamide with an aromatic substituent enhanced the substrate potential for liver NAT, III may be useful as a substrate for the rapid classification of slow and fast acetylators.

4-Aminobenzoic Acid↗
PubMed1989

Trametes versicolor ligninase: isozyme sequence homology and substrate specificity.

The substrate specificity of three ligninase isozymes from the white-rot fungus Trametes versicolor has been investigated using stereochemically defined synthetic dimeric models for lignin. The isozymes have been found to attack non-phenolic beta-O-4 as well as beta-1 lignin model compounds. This finding confirms the classification of the isozymes from T. versicolor as ligninases. The amino-terminal residues of the three isozymes from T. versicolor have been determined using Edman degradation. Minor differences found between the sequences suggest the existence of several structural genes for ligninase in T versicolor. Comparisons have been made with the sequences of three previously reported ligninases from Phanerocompaete chrysosporium, another lignin-degrading fungus. One of the sequences from P. chrysosporium is distinctly more similar to the T. versicolor isozymes than to the other two sequences from P. chrysosporium.

Amino Acid Sequence↗