ThetrpB8 mutation of Escherichia coli tryptophan synthase
Abstract
The trpB8 mutation of E. coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation. A novel phenotype of trpB8, namely the ability to respond nutritionally to L-serine, was examined genetically and physiologically. The trpB8 allele was cloned and sequenced. The G to C transversion at nucleotide 5528 of the trp operon resulted in a switch at codon 281 of the $\beta$ subunit from Gly to Arg. G281 is within the proteolytically sensitive "hinge" region and is invariant among all the $\beta$ polypeptides that have been structurally characterized. The cloned $\beta$(B8) subunit was partially purified. Under certain assay conditions, $\beta$(B8) exhibits $\beta$-reaction activity. The pH optimum for the mutant enzyme was 9.8, which is 2 units higher than that of wild type. Ammonium ion effectively stimulated the activity. $\beta$(B8) subunit exists in the form of a dimer. The catalytic activity of $\beta\sb2$(B8) was stimulated by $\alpha$ subunit. No $\alpha$-$\beta\sb2$(B8) complex could be physically detected in vitro. A volume excluder, PEG6000, was capable of enhancing the $\alpha$ subunit stimulatory effect. An absorption spectroscopic study indicated that the catalytic lesion caused by the G281R mutation is a block in the conversion of an enzyme-substrate Schiff base intermediate (ESII) into other reaction intermediates. Fluorescence spectroscopy further indicated that this intermediate is probably more solvent-exposed than that of the wild type enzyme. Three lines of evidence suggest that in comparison to wild type, $\beta$(B8) subunit has an altered conformation. $\beta$(B8) aggregates within cells upon overexpression. The mutant protein is susceptible to cleavage by OmpT protease. The OmpT cleaved N-terminal fragment is able to interact with a "hinge" region-specific monoclonal antibody that is unreactive toward the intact $\beta$(B8) polypeptide. The physicochemical behavior of $\beta\sb2$(B8) protein is supported by computer graphic modelling studies. An open vs. closed model of conformational change within the $\beta\sb2$ protein during ligand binding, subunit interaction and catalysis is proposed. The "hinge" region is believed to have an essential role in controlling this process and maintaining the active (closed) conformation.
Degree
Ph.D.
Advisors
Somerville, Purdue University.
Subject Area
Biochemistry
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