Limited proteolysis of N-(5′-phosphoribosyl)anthranilate isomerase: Indoleglycerol phosphate synthase from Escherichia coli yields two different enzymically active, functional domains

Abstract

The native enzyme must be denatured either by sodium dodecyl sulfate or by urea before limited proteolysis can occur. Under these conditions only one or two peptide bonds are hydrolyzed by each of the following proteases: Staphylococcal V8 protease, trypsin and elastase. The amino-terminal amino acid sequences were determined to identify the cleavage sites. The new sequences comprise approximately 20% of the entire polypeptide chain, and show good agreement with the nucleotide sequence of the trpC gene. Both V8 protease † † Abbreviations used: V8 protease, extracellular protease from Staphylococcus aureus strain V8: PRA, N-(5′-phosphoribosyl)anthranilate; InGP, indoleglycerol phosphate; CdRP, 1-(2-carboxyphenyl-amino)-1-deoxyribulose 5-phosphate; SDS, sodium dodeoyl sulfate. and elastase yield large carboxy-terminal fragments, about two thirds of the size of the parent enzyme, and corresponding small amino-terminal fragments. Trypsin cleaves a single peptide bond in the last one third of the polypeptide chain. After separation of the fragments, removal of dodecyl sulfate and renaturation, only the large fragments fold to stable structures. The small fragments precipitate. The large amino-terminal fragment catalyzes only the synthesis of indoleglycerol phosphate and precipitates when solutions are frozen and thawed. The large carboxy-terminal fragment catalyzes only the isomerization of N-(5′-phosphoribosyl)anthranilate and is stable towards freezing and thawing. These studies prove that the intact bifunctional enzyme consists of two autonomously folding, functional domains. They also support the notion that the bifunctional enzyme may have arisen by the fusion of separate ancestral genes, and that stabilization of the intrinsically labile indoleglycerol phosphate synthase domain by interdomain interactions is functionally advantageous.