Structural and functional studies of serine acetyltransferase

• Main Author: Hindson, Vincent John
• Published: University of Leicester 1995
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.674402

Recombinant serine acetyltransferase was overexpressed in Escherichia coli and purified 13-fold to homogeneity. The specific activity of the purified enzyme was 719 units/mg. Hydrodynamic and quasi-elastic light scattering studies indicated that SAT has an open hexameric quaternary structure, furthermore, chemical cross-linking studies indicated that the fundamental building block is a trimer. Steady-state and stopped-flow kinetic studies indicated that the forward reaction proceeds via a ternary complex, and dead-end inhibition analyses indicated that the order of substrate addition was random. Substrate inhibition by serine indicated that the breakdown of the SAT-CoA complex is partially rate-determining, whereas the linearity of primary double-reciprocal plots and inhibition replots suggested that the interconversion of ternary complexes is not significantly faster than kcat. Hence the kinetic data is consistent with a steady-state random-order reaction mechanism in which the interconversion of ternary complexes and the dissociation of CoA may be both partially rate-determining. Steady-state kinetic studies with the alternative acyl acceptor threonine were consistent with such a mechanism, whereas, those with propionyl CoA satisfied the requirements of a rapid-equilibrium random-order reaction mechanism. Steady-state kinetic studies of the reverse reaction were consistent with a steady-state random-order reaction mechanism for SAT in which the breakdown of the enzyme-serine complex is partially rate-determining. Calorimetric titration data indicated that cysteine does not bind at the coenzyme binding site and may not bind at the serine binding site; hence it may bind at an allosteric site. The pH dependence of kcat/Km suggested that a base, with a pK in the region of 6, might have a role in catalysis. Chemical modification of all three cysteines in SAT by [14C]- iodoacetamide demonstrated that the enzyme exists in the fully reduced state. Furthermore, SAT was irreversibly inhibited by iodoacetamide and substrates conferred protection against such inhibition. Silica thin layer eletrophoresis demonstrated the labelling of both histidine and cysteine in native and denatured preparations of [14C]-iodoacetamide-modified SAT. Peptide sequencing was employed to further characterise the residues labelled in the native state.