Biomedical subjects

R E Viola

Publications and source records attributed to R E Viola.

At least 37 records · Page 2Linked to original sources

PubMed1996

Conversion of cysteinyl residues to unnatural amino acid analogs. Examination in a model system.

Improved and efficient techniques have led to an explosive growth in the application of site-directed mutagenesis to the study of enzymes. However, the limited availability of only those 20 amino acids that are translated by the genetic code has prevented the systematic variation of an amino acid's properties in order to define more precisely its role in the catalytic mechanism of an enzyme. An approach is being examined that combines the high specificity of site-directed mutagenesis with the flexibility of chemical modification to overcome these limitations. A set of reagents has been synthesized and reacted with a cysteine model to produce a series of amino acid structural analogs at appreciable rates and in good overall yields. The selective incorporation of these analogs in place of important functional amino acids in a protein will allow a more detailed examination of the role of that amino acid.

Amino Acids↗

PubMed1995

Use of structural comparisons to select mutagenic targets in aspartate-beta-semialdehyde dehydrogenase.

L-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) from Escherichia coli has been probed by site-directed mutagenesis to identify residues that play an important function in the catalytic activity of the enzyme. Sequence homology searching among ASA DHs that have been isolated from other species and comparisons with the structures of functionally similar D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) that have been solved from several species have been utilized to select appropriate targets for mutagenesis. A highly conserved active site glutamine has been identified in the E. coli ASA DH that enhances the reactivity of the enzyme. Alteration of this residue leads to an enzyme with reduced catalytic efficiency, yet with an unchanged binding affinity for substrates and coenzyme. Replacement of an arginine residue that is conserved throughout the ASA DH and GAPDH enzyme families leads to a significant decrease in catalytic turnover and is the only mutation examined that also results in a decreased affinity for the substrates of the reaction. This residue is assigned a role in the binding of the substrate aspartate-beta-semialdehyde. Sequence alignment of ASA DH with other NADP- and NAD-dependent enzymes has resulted in the identification of a putative pyridine nucleotide binding region. Substitution of two amino acids in this region with neutral or positively charged side chains has resulted in a change in enzyme specificity. For wild-type ASA DH, NADP is strongly favored as the coenzyme, while in this mutated enzyme the selectivity has been lowered by a factor of 60, and this enzyme has comparable affinities for either pyridine nucleotide.

Amino Acid Sequence↗

PubMed1995

Elimination of the sensitivity of L-aspartase to active-site-directed inactivation without alteration of catalytic activity.

The catalytic activity of the enzyme L-aspartase from Escherichia coli has previously been shown to be sensitive to sulfhydryl reagents. The use of group-specific reagents, and a sequence homology comparison study among the fumarase-aspartase family of enzymes, has not, however, lead to the identification of a specific, essential cysteinyl residue. We have recently shown that L-aspartate-beta-semialdehyde is an alternative substrate for L-aspartase, producing fumaric acid semialdehyde (FAA) which specifically inactivates the enzyme [Schindler, J. F., & Viola, R. E. (1994) Biochemistry 33, 9365]. Proteolytic digests of the resulting inactivated enzyme have now been mapped by HPLC and mass spectrometry. A specific residue (Cys-273) has been determined to be the site of FAA modification. Site-directed mutagenesis of this cysteine in the E. coli enzyme has produced altered enzymes which are considerably less sensitive to active-site-directed inactivation, while retaining full catalytic activity. Thus, cysteine-273 has been identified as an active-site nucleophile that, while not directly involved in catalysis in L-aspartase, is poised to attack an activated double bond in an enzyme-bound product analogue.

Amino Acid Sequence↗

PubMed1994

Mechanism-based inactivation of L-aspartase from Escherichia coli.

The substrate analogue L-aspartate beta-semialdehyde (L-ASA) has been identified as a mechanism-based inactivator of L-aspartase from Escherichia coli. The enzyme catalyzes the deamination of L-ASA to yield fumaric acid semialdehyde (FAA) and NH4+, with the product FAA partitioning between subsequent release or irreversible enzyme inactivation. Complete protection against L-ASA inactivation is observed in the presence of the product fumarate and a divalent metal ion. However, protection against inactivation by the product FAA also requires the presence of an enzyme activator. In addition to functioning as a mechanism-based inactivator, L-ASA has also been shown to serve as an activator of L-aspartase. The mechanism of inactivation by FAA involves the attack of an active site nucleophilic at the alpha-carbon of FAA to yield a stable Michael type enzyme adduct. Subsequent formation of a hydrazone upon treatment of the enzyme adduct with 2,4-dinitrophenylhydrazine confirms the presence of the unreacted aldehydic group of FAA. Examination of a group of product analogues with different substituents has demonstrated a correlation between the electron-withdrawing ability of these functional groups and the rate of inactivation of L-aspartase.

Aspartate Ammonia-Lyase↗

PubMed1994

Mutagenic investigation of conserved functional amino acids in Escherichia coli L-aspartase.

The potential importance of several functional amino acids in the activity of L-aspartase from Escherichia coli has been examined by site-directed mutagenesis. Amino acids whose importance in enzyme activity was suggested by chemical modification and pH dependence studies were chosen as candidates for investigation. The selection of the particular amino acid targets was guided by homology comparisons among the other sequenced bacterial L-aspartases and by the broader comparison among the fumarase-aspartase enzyme family. Substitution of the most highly conserved cysteine with either serine or alanine, or the most highly conserved histidine with leucine, had no significant effect on the activity of L-aspartase or on the sensitivity of these mutated L-aspartases to cysteine and histidine specific modifying reagents. However, alteration of each of the two conserved lysines to arginine did cause dramatic changes in the catalytic properties of the enzyme. Modification of lysine 54 results in the complete loss of enzyme activity. However, this activity loss appears to be related to changes in the subunit association properties of the arginine 54 mutant. Lysine 326 appears to be involved in substrate binding. Modification of this residue causes a 5-fold increase in the Km for aspartic acid, a drastic decrease in kcat/Km, and a change in the divalent metal ion requirements of the enzyme.

Amino Acid Sequence↗

PubMed1994

Inactivation of yeast alcohol dehydrogenase by nitrilopropionamides.

A series of halonitrilopropionamides have been examined as potential inhibitors of yeast alcohol dehydrogenase. Analogues with a good leaving group on the alpha-carbon, and a geminal electronegative atom, were found to be initial competitive inhibitors against NAD with inhibition constants as low as 0.6 microM. Incubation of the enzyme with these inhibitors leads to a slow, irreversible inactivation, with an inactivation constant for 2,2-dibromo-3-nitropropionamide of about 100 microM. No protection against inactivation was observed with the substrate ethanol, while the presence of saturating levels of NAD slowed the rate, but not the final extent, of enzyme inactivation. The resulting enzyme-inactivator complex is stable to a range of conditions that are known to denature the enzyme, indicating that a covalent modification of yeast alcohol dehydrogenase has led to the inactivation. A bimodal inactivation model is proposed to account for the observed interactions of these halonitrilopropionamides with yeast alcohol dehydrogenase.

Alcohol Dehydrogenase↗

PubMed1993

Crystallization and preliminary X-ray studies of L-aspartase from Escherichia coli.

Single crystals of L-aspartate ammonia-lyase (L-aspartase) from Escherichia coli have been obtained by microdialysis at room temperature using polyethylene glycol 3350 and sodium acetate as co-precipitants. The crystals exhibit the symmetry of space group P2(1)2(1)2 with a = 156.5 A, b = 147.6 A, c = 102.5 A and diffract at least to 2.8 A.

Aspartate Ammonia-Lyase↗

PubMed1992

Crystallization and preliminary crystallographic analysis of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

Aspartate-beta-semialdehyde dehydrogenase catalyzes the NADPH-mediated reductive dephosphorylation of beta-aspartylphosphate at a branch point in the biosynthesis of several amino acids. The enzyme from Escherichia coli has been crystallized by the vapor diffusion method from Tris buffer (pH 8.5) using polyethylene glycol 4000 as a precipitant. The crystals are orthorhombic and have the symmetry of space group P222(1), with unit cell dimensions of a = 177.8 A, b = 59.9 A, c = 118.65 A, and alpha = beta = gamma = 90 degrees. The dimensions and space group are indicative of two enzyme dimers (40 kDa per subunit) in the asymmetric unit. The crystals show strong diffraction, and a native data set has been collected to 2.5 A resolution.

Aspartate-Semialdehyde Dehydrogenase↗

PubMed1992

Identification of an essential cysteine in the reaction catalyzed by aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The enzyme L-aspartate-beta-semialdehyde dehydrogenase from Escherichia coli has been studied by oligonucleotide-directed mutagenesis. The focus of this investigation was to examine the role of a cysteine residue that had been previously identified by chemical modification with an active site directed reagent (Biellmann et al. (1980) Eur. J. Biochem. 104, 59-64). Substitution of this cysteine at position 135 with an alanine results in complete loss of enzyme activity. However, changing this cysteine to a serine yields a mutant enzyme with a maximum velocity that is 0.3% that of the native enzyme. This C135S mutant has retained essentially the same affinity for substrates as the native enzyme, and the same overall conformation as reflected in identical behavior on gel electrophoresis and in identical fluorescence spectra. The pH profile of the native enzyme shows a loss in catalytic activity upon protonation of a group with a pKa value of 7.7. The same activity loss is observed at this pH with the serine-135 mutant, despite the differences in the pKa values for a cysteine sulfhydryl and a serine hydroxyl group that have been measured in model compounds. This observed pKa value may reflect the protonation of an auxiliary catalyst that enhances the reactivity of the active site cysteine nucleophile in the native aspartate-beta-semialdehyde dehydrogenase.

Amino Acid Sequence↗

PubMed1992

Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli.

The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.

Aspartate Kinase↗

PubMed1991

Kinetic studies of L-aspartase from Escherichia coli: pH-dependent activity changes.

The pH dependence of the kinetic parameters of the L-aspartase-catalyzed reaction have been examined in both the amination and the deamination directions. The enzyme isolated from Escherichia coli exists in a pH-dependent equilibrium between a higher pH form that has an absolute requirement for a divalent metal ion and for substrate activation, and a low pH form that does not require activation by either substrate or metal ions. The interconversion between these enzyme forms is observed near neutral pH in the profiles examined for the reaction in either direction. This pH-dependent activation has not been observed for other bacterial aspartases. Loss of activity is observed at high pH with a pK value of 9. The pH profiles of competitive inhibitors such as 3-nitropropionic acid and succinic acid have shown that the enzyme group responsible for this activity loss must be protonated for substrate binding at the active site. An enzymatic group has also been identified that must be protonated in the amination reaction, with a pK value near 6.5, and deprotonated in the deamination reaction. This group, tentatively assigned as a histidyl residue, fulfills the criteria for the acid-base catalyst at the active site of L-aspartase.

Amination↗

PubMed1991

Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.

Aspartate-Semialdehyde Dehydrogenase↗

PubMed1990

The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli.

The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined. Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+. Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor. Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme. The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism. Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.

Adenosine Diphosphate↗

PubMed1989

Aluminum-adenine nucleotides as alternate substrates for creatine kinase.

Aluminum-ATP has previously been shown to be a potent but selective inhibitor of certain kinases (N. C. Furumo and R. E. Viola (1989) Inorg. Chem. 28, 820-823). Because of the selective binding affinity that has been demonstrated, aluminum-ATP was examined as a potential alternate substrate for enzyme-catalyzed phosphoryl-transfer reactions. Of the kinases that have been examined only creatine kinase, which was weakly inhibited by Al-ATP, was found to utilize this metal-nucleotide complex as a substrate. The maximum velocity for this reaction is 0.6% of that observed with Mg-ATP, and a Michaelis constant of 90 microM was measured, which is comparable to that with Mg-ATP. The equilibrium constant for the reaction, measured by phosphorus-31 NMR spectroscopy, is 0.18. This value is indistinguishable from the value determined for the reaction with Mg-ATP as the phosphoryl donor. The rate of interconversion between reactants and products at stoichiometric levels of creatine kinase was found to be quite rapid in the presence of Al3+, indicating that the rate-limiting step in catalytic turnover for this substrate must be a slow rate of substrate binding and/or product release.

Adenosine Diphosphate↗

PubMed1989

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity.

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.

Alcohol Oxidoreductases↗

PubMed1988

L-aspartase from Escherichia coli: substrate specificity and role of divalent metal ions.

The enzyme L-aspartase from Escherichia coli has an absolute specificity for its amino acid substrate. An examination of a wide range of structural analogues of L-aspartic acid did not uncover any alternate substrates for this enzyme. A large number of competitive inhibitors of the enzyme have been characterized, with inhibition constants ranging over 2 orders of magnitude. A divalent metal ion is required for enzyme activity above pH 7, and this requirement is met by many transition and alkali earth metals. The binding stoichiometry has been established to be one metal ion bound per subunit. Paramagnetic relaxation studies have shown that the divalent metal ion binds at the recently discovered activator site on L-aspartase and not at the enzyme active site. Enzyme activators are bound within 5 A of the enzyme-bound divalent metal ion. The activator site is remote from the active site of the enzyme, since the relaxation of inhibitors that bind at the active site is not affected by paramagnetic metal ions bound at the activator site.

Ammonia-Lyases↗

PubMed1986

Kinetic studies of L-aspartase from Escherichia coli: substrate activation.

The enzyme L-aspartase from Escherichia coli was observed to have a time lag during the production of aspartic acid from fumarate and ammonia. This time lag is pH dependent, with little lag observed below pH 7.0 and a very extensive lag observed above pH 8.0. This time lag was also found to be dependent on both substrate and divalent metal ion concentrations and on the degree of proteolysis of L-aspartase. The observed lag, in the reaction examined in the amination direction, has been found to be correlated with the nonlinear kinetics seen at higher pH in the deamination direction. Both phenomena are consistent with a model in which there is a separate activator site for the substrate, L-aspartic acid, that is distinct from the enzyme active site. Occupation of this site by the substrate, or by various substrate analogues, eliminates both the nonlinearity and the time lag. The D isomer of aspartic acid, which does not bind at the active site, can bind at this newly identified activator site.

Ammonia-Lyases↗

PubMed1985

Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography.

Improved purification schemes are reported for the enzymes L-aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli. Dye-ligand chromatography on commercially available dye matrices are incorporated as key steps in these purifications. Red A-agarose has a high affinity for L-aspartase, which is then eluted as a homogeneous protein fraction with 1 mM L-aspartic acid. Green A-agarose shows a high binding affinity for the bifunctional enzyme aspartokinase-homoserine dehydrogenase I. Purification is accomplished by elution with NADP+, followed by formation of a ternary complex with NADP and cysteine, a good competitive inhibitor of the homoserine dehydrogenase activity, and rechromatography on Green A-agarose. The final specific activity of each purified enzyme equaled or exceeded previously reported values, the overall yield of enzymes obtained was significantly higher, and these improved purification schemes were found to be more amenable to being scaled up for the production of large quantities of purified enzyme.

Ammonia-Lyases↗