Abstract
The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.
Highlights
The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH
From this we have argued that NADH inhibition in the Type II enzymes is an evolutionary add-on; Type I dimers were altered by a series of mutations to generate NADH sites and new contact surfaces so that hexamers could form
Kinetic Properties of Protein Variants with Amino Acid Replacements in the NADH Binding Site—The structure of the citrate synthases (CS)-NADH complex allows the prediction of a number of hydrogen bonds between NADH and the protein, including nine involving amino acid side chains [4]
Summary
Preparation and Characterization of Variant Proteins—Variant CS proteins were prepared by site-directed mutagenesis, using the QuikChangeTM kit from Stratagene. Rate data were collected over a range of concentrations of the two substrates, acetyl-CoA and OAA, in buffer containing 0.1 M KCl, in which wild type E. coli CS obeys the ordered bisubstrate steady-state mechanism, with OAA binding first These data were fitted to the appropriate steady-state equation as described [8]. The space group and unit cell parameters obtained (Table I) indicated that crystals of both the R109L variant and the R109L-NADH complex were isomorphous with those of wild type E. coli CS [3]. This permitted the wild type structure (with residue 109 truncated to an alanine) to be used as the starting refinement model for the R109L variant protein. An assessment of the final refined structures obtained indicates that both of these have excellent polypeptide chain geometry (Table I)
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