STRUCTURE-FUNCTION RELATIONSHIPS OF GLUTAMINE AMIDOTRANSFERASES

J. YUN TSO, Purdue University

Abstract

Two bacterial glutamine amidotransferases, Serratia marcescens anthranilate synthase and Escherichia coli glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase), have been studied to examine the reaction mechanism and structure-function relationships of glutamine amidotransferases. The amino acid sequence of anthranilate synthase Component II (AS II) from S. marcescens was determined. The cysteine residue essential for glutamine utilization was alkylated selectively by iodo{1-('14)C}acetamide prior to separation of two protein components of anthranilate synthase. The isolated AS II was then subjected to cleavage by cyanogen bromide and by trypsin after citraconylation to obtain overlapping fragments. AS II is a single polypeptide chain of 192 residues with a calculated molecular weight of 20,956. Cysteine-83 was identified as the essential residue. The active site region is virtually identical to that of Pseudomonas putida AS II. L-((alpha)S,5S)-(alpha)-Amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) an antitumor drug, is an active site-directed affinity analog of glutamine. It selectively inactivated the glutamine dependent activity of S. marcescens anthranilate synthase. A reversible noncovalent complex was formed prior to irreversible enzyme modification. Inactivation of anthranilate synthase resulted from incorporation of one equivalent of AT-125 per enzyme protomer. Active site cysteine-83 in S. marcescens AS II was the residue alkylated by AT-125. Residues essential for S. marcescens anthranilate synthase Component I (AS I) were studied by chemical modification reactions. Phenylglyoxal and 1,2-cyclohexanedione modified 2-5 arginine residues and inactivated AS I. The substrate chorismate reduced the rate of inactivation. Analysis of inactivation data indicated that one arginine residue is essential for activity. Histidine residues in AS I were modified by ethoxyformic anhydride and by photooxidation. Enzyme inactivation accompanied modification of histidine residues. Inactivation was prevented by substrate. Comparison of the number of carbethoxy groups incorporated between substrate-protected and unprotected AS I indicated that one histidine residue is required for activity. AS I was also inactivated by bromopyruvate. Substrate retarded inactivation by bromopyruvate. A differential labeling experiment indicated that the loss of AS I activity was correlated with alkylation of one cysteine residue. A tryptic peptide containing the essential cysteine residue was isolated. The E. coli purF gene encodes the enzyme catalyzing the first reaction in purine biosynthesis, amidophosphoribosyltransferase. purF was subcloned from a ColEl-purF plasmid into plasmid pBR322 and was localized in a 3.1 kb restriction fragment. A detailed restriction map of this fragment was determined. E. coli strains harboring hybrid plasmids overproduced amidophosphoribosyltransferase. Purified amidophosphoribosyltransferase lacks iron as well as other trace metals as judged by X-ray fluorescence spectrometry. The NH(,2)-terminal and COOH-terminal amino acid sequences of amidophosphoribosyltransferase were determined. {6-('14)C}-Diazo-5-oxo-norleucine (DON), an active site-directed affinity analog of glutamine, selectively inactivated the glutamine-dependent amidophosphoribosyltransferase activity. Inactivation of amidophosphoribosyltransferase was accompanied by incorporation of one equivalent of {6-('14)C}DON per enzyme protomer. A partial amino acid sequence of a tryptic peptide labeled by {6-('14)C}DON was determined. Comparison of the glutamine site of Escherichia coli amidophosphoribosyltransferase with glutamine sites from Serratia marcescens anthranilate synthase, E. coli GMP synthetase and chicken liver formylglycinamide ribonucleotide amidotransferase revealed no sequence homology.

Degree

Ph.D.

Subject Area

Microbiology

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