A cysteine-histidine-aspartate catalytic triad is involved in glutamine amide transfer function in purF-type glutamine amidotransferases

B Mei,H Zalkin

doi:10.1016/s0021-9258(19)84750-6

Abstract

A family of four glutamine amidotransferases has a homologous glutamine amide transfer domain, designated purF-type, that is named after purF-encoded glutamine phosphoribosylpyrophosphate amidotransferase. The glutamine amide transfer domain of approximately 194 amino acid residues is at the NH2 terminus of the protein chain. Site-directed mutagenesis was used to replace several of the 9 invariant amino acids in the glutamine amide transfer domain of glutamine phosphoribosylpyrophosphate amidotransferase. The results indicate that a Cys1-His101-Asp29 catalytic triad is involved in the glutamine amide transfer function of this enzyme. The evidence suggests that His101 functions to increase the nucleophilicity of Cys1, which is used to form a glutamine-enzyme covalent intermediate. Asp29 has a role subsequent to formation of the covalent intermediate. The Cys-His-Asp catalytic triad is implicated in the glutamine amide transfer function of purF-type amidotransferases.

Highlights

A family of four glutamine amidotransferases has a transfer domain
We have investigated purF-encoded amidophosnicotinamide coenzymes, and the amino sugar, glucosamine phoribosyltransferase
ThepurF-type GlutamineAmide Transfer Domain-Amino acid sequences have been derived for several amidotransferases that constitute an apparently homologous purF-type subfamily

Summary

Introduction

A family of four glutamine amidotransferases has a transfer domain. One amidotransferase subfamily designated homologous glutamine amide transfer domain, desig- trpG-type [8]includes anthranilate synthase [15, 16],p-aminated purF-type, thrat is named after purF-encoded nobenzoate synthase [17, 18],carbamoyl-phosphate syntheglutamine phosphoribosylpyrophosphate amidotrans- tase [19, 20], CTP synthetase [7], formylglycinamide riboferase. The Cys-His-Asp imsarum [28] This subfamily has been designatedpurF-type catalytic triad is implicated in the glutamine amide [8] after the prototype purl”-encoded amidophosphoribosyltransfer function of purF-type amidotransferases. Alignments of pairs of sequences have been given [27, 28] but not a compilation of all four sequences In these enzymes, the essential cysteine required for formation of the covalent glutaminyl intermediate is the NHs-terminal residue. For each of the enzymes, an role of histidine and aspartate residues in addition to the NHa-dependent activity of the type R f NHs + RNH, has essential cysteine inthe glutamine amide transfer function of been demonstrated or inferred in addition to the glutamine- amidophosphoribosyltransferase and implicates these resi-

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