Abstract
D-3-Phosphoglycerate dehydrogenase from Escherichia coli is a homotetrameric enzyme which is allosterically regulated by the end product of its pathway, L-serine. The enzyme binds 4 L-serine molecules at two interfaces formed by the noncovalent association of the regulatory domains. The two domains that comprise each interface are related by an approximately 180 degrees axis of symmetry, and two serine molecules bind at each interface by forming a hydrogen bond network between the domains. A model has been proposed that suggests that serine functions by drawing adjacent domains together and that this in turn translates a conformational change to the active site. A tryptophan residue has been engineered into the helices flanking the regulatory interfaces that displays significant quenching in response to serine binding. Residues on the adjacent subunit appear to be primarily responsible for the tryptophan quenching and thus support the hypothesis that serine binding leads to an increase in the proximity between residues on neighboring subunits. Serine binding studies show that this quenching, as well as inhibition of enzymatic activity, are essentially complete when only two of the four serine binding sites are occupied. The requirement for only one serine per interface is consistent with the notion that the interface is formed by relatively rigid domains and that hydrogen bonding at only a single site is all that is required to substantially close the interface. The fluorescence quenching in response to L-serine binding generally correlates with enzymatic inhibition, but there appears to be a slight lag in inhibition relative to quenching at low serine concentrations. The observed fluorescence quenching of residues in the regulatory domains of D-3-phosphoglycerate dehydrogenase provide the first direct evidence for a conformational change in response to effector binding and provide a means to monitor the first step in the allosteric mechanism.
Highlights
D-3-Phosphoglycerate dehydrogenase (PGDH)1 (EC 1.1.1.95) from Escherichia coli is an allosterically regulated enzyme that is inhibited by L-serine, the end product of its metabolic pathway [1, 2]
The mutations at E360W and D388W place tryptophan residues on each of the two ␣-helices that run parallel to the regulatory domain interface
E360W is in the helix bordering the domain interface, whereas D388W is in the outer helix approximately 17 Å from the interface
Summary
D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) from Escherichia coli is an allosterically regulated enzyme that is inhibited by L-serine, the end product of its metabolic pathway [1, 2]. Earlier work [5] demonstrated that tethering adjacent regulatory domains together by engineering disulfide bonds across the interface produced an inhibited enzyme in the absence of L-serine. This inhibitory effect was reversible by reduction and could be repeated through successive cycles of oxidation and reduction of the disulfide bonds. These observations led to the hypothesis that the result of serine binding was to draw the adjacent regulatory domains together, because covalently coupling them led to a qualitatively similar result. The work reported here describes tryptophan fluorescence studies consistent with this hypothesis
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