Abstract
The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.
Highlights
The Escherichia coli pyruvate dehydrogenase complex (PDHc)1 plays a pivotal role in carbohydrate utilization by bacterial cells according to the Reaction 1
Peptide maps were generated for PDHc-E1 and its E636A variant in complex with thiamine diphosphate (ThDP) according to our protocol presented in this paper and reported recently (Fig. 2, A, B, and C) [12]
The following regions of interest were compared in the two peptide maps: peptides encompassing amino acid residues 1–55, 401– 413, and 541–557, for which no electron density was found in the crystal structure of PDHc-E1 [2]; the peptide 157–181 encompassing the amino acid residues Tyr177 and His179, which was reported earlier to have a role in stabilization of reaction intermediates [8]; and the peptide 240 –247 encompassing amino acid residues from the ThDP binding fold first identified by sequence alignments of ThDP enzymes (Gly229–Asn260) [13]
Summary
Materials—The QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Spectrometry; HEThDP, C2␣-hydroxyethylthiamine diphosphate; LThDP, C2␣-lactylthiamine diphosphate; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; PDHc-E1, the first ThDP-dependent subunit of PDHc; 1-lip PDHc-E2, the second subunit of the complex, in this paper carrying a single lipoyl domain; PDHc-E2-E3 subcomplex, complex formed between the two subunits and used for the overall activity measurements; PLThDP, phosphonolactylthiamine diphosphate; ThDP, thiamine diphosphate; ThTDP, thiamine 2-thiazolone diphosphate; YPDC, yeast pyruvate decarboxylase. Identification of these peptides was accomplished by matching their monoisotopic signals from the deconvoluted mass spectra to the theoretical monoisotopic values of the expected tryptic peptides using the Sequence Editor of Bruker’s biotool program, version 2.0
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