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E R Kantrowitz

Publications and source records attributed to E R Kantrowitz.

117 records · Page 7Linked to original sources
PubMed1980

An essential arginine residue in porcine phospholipiase A2.

Reaction of phenylglyoxal, a reagent specific for arginine residues, with porcine phospholipiase A2 results in complete elimination of catalytic activity. The modification reaction is markedly dependent on pH. Other dicarbonyl compounds such as 2,3-butanedione and 1,2-cyclohexanedione also react with the enzyme to cause loss of activity but at significantly slower rates. At pH 7.0, the inactivation can be slightly retarded in the presence of Ca2+ and almost completely prevented in the presence of n-alkylphosphorylchline inhibitors. At pH 8.5, the n-alkylphosphorylcholine inhibitors are less effective. The decrease in enzymatic activity correlates with the modification of about 1 arginine residue/phospholipase A2 molecule. The data suggest that the arginine is involved in the binding of the phosphate portion of the substrate to the enzyme.

Aldehydes↗
PubMed1980

The role of tryptophan in aspartate transcarbamylase.

Replacement of 7-azatryptophan for tryptophan in two positions on the catalytic chain of aspartate transcarbamylase results in changes in the enzyme's homotropic and heterotropic interactions although there is no change in the enzyme's specific activity. The extent of azatryptophan incorporation was quantitated by amino acid analysis which showed that 85% of the tryptophan residues had been replaced. The substituted enzyme is activated by ATP and inhibited by CTP to a greater extent than is the native enzyme. The aspartate saturation curve in the presence of ATP is identical for the two enzymes, but the curve in the presence of CTP and without effectors is shifted toward higher aspartate concentrations for the azatryptophan-substituted enzyme. At low aspartate concentrations, the native enzyme is activated to a greater extent by the substrate analog succinate. These data suggest that the substitution renders the low substrate affinity conformational state of the enzyme less catalytically efficient. This interpretation is in agreement with possible side chain interactions observed in the three-dimensional structure of the enzyme.

Aspartate Carbamoyltransferase↗
PubMed1980

Isolation and preliminary characterization of single amino acid substitution mutants of aspartate carbamoyltransferase.

In order to isolate functional Escherichia coli aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC2.1.3.2) with single amino acid replacements, a series of pyrB nonsense mutants has been isolated. These nonsense mutants were induced by 2-aminopurine mutagenesis and selected by a combination of antibiotic treatments, direct enzyme assays, and suppressibility tests. Suppression of the pyrB nonsense mutation with various suppressors, which insert different amino acids, has resulted in the formation of a series of mutant aspartate carbamoyltransferases, each differing in one amino acid from the wild-type enzyme. After partial purification, kinetic studies revealed that some of the mutant enzymes had altered homotropic and heterotropic interactions. The mutants that had a tyrosine insert showed the most pronounced changes, followed by those with a serine insert. The mutants having a glutamine insert, howevr, were indistinguishable from the wild-type enzyme, supporting the conclusion that, because of the specificity of the mutagen, the glutamine insert had regenerated the wild-type enzyme.

2-Aminopurine↗
PubMed1977

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.

Aldehydes↗
PubMed1977

Interaction of tetraiodofluorescein with a modified form of aspartate transcarbamylase.

Low concentrations of the dye tetraiodofluorescein activate native aspartate transcarbamylase (aspartate carbomoyltransferase, carbomoylphosphate:L-aspartate carbomoyltransferase, EC 2.1.3.2), while high concentrations inhibit the enzyme's activity [Jacobsberg, L. B., Kantrowitz, E. R. & Lipscomb, W. N. (1975) J. Biol. Chem. 250, 9238-9249]. This dye is now shown to produce similar effects upon a modified form of aspartate transcarbamylase produced by Escherichia coli grown in a culture medium supplemented with thiouracil. Significantly, the ATP-induced activation is reduced in the modified form of the enzyme to the same extent as is the tetraiodofluorescein-induced activation. Thus, a relationship is demonstrated between the internal mechanisms by which ATP and tetraiodofluorescein activate aspartate transcarbamylase.

Adenosine Triphosphate↗
PubMed1976

An essential residue at the active site of aspartate transcarbamylase.

Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both.

Amino Acids↗
PubMed1975

Interaction of tetraiodofluorescein with aspartate transcarbamylase and its isolated catalytic and regulatory subunits.

The interaction of the dye tetraiodofluorescein with native aspartate transcarbamylase and its isolated subunits has been investigated by both binding and activity measurements at 4 and 23 degrees. At room temperature low concentrations of tetraiodofluorescein activate the native enzyme, but high concentrations inhibit the enzyme's activity. At the low temperature the native enzyme is inhibited by all concentrations of dye. Isolated catalytic subunit is very effectively inhibited at both temperatures. For the native enzyme these results are explained by 18 tetraiodofluorescein sites of approximately equal affinity (K = 7.3 X 10(-5) M) on each enzyme hexamer: one class of six sites at the nucleoside triphosphate effector binding sites is responsible for the activation, a second class which competes with the substrate carbamylphosphate causes the inhibition, and a third class does not interact with either the effectors or the substrates. Measurements of tetraiodofluorescein binding to isolated regulatory, catalytic, and p-hydroxymercuribenzoate-inactivated catalytic subunits support the above assignments. This scheme of tetraiodofluorescein binding sites successfully predicts the changes in the tetraiodofluorescein-aspartate transcarbamylase difference spectrum induced by the addition of various ligands. The activity changes induced by the dye are explained if the binding of a single tetraiodofluorescein molecule to one of the six regulatory sites activates all six of the catalytic sites, while while a dye molecule binding to any one of the catalytic sites inactivates only that catalytic site.

Aspartate Carbamoyltransferase↗