Studies on the enzymatic cis dehydration of 3-dehydroquinic acid catalyzed by escherichia coli 3-dehydroquinate dehydratase
Description
3-Dehydroquinase has been purified to apparent electrophoretic homogeniety from Escherichia coli 83 2. The enzyme exists as a tetramer with a minimum molecular weight of 40,000 daltons. To account for the cis stereochemistry of the enzymatic dehydration catalyzed by 3-dehydroquinase, it is proposed that the enzyme induces a twist boat conformational change in 3-dehydroquinic acid that moves the carboxylate to a pseudo axial configuration in the enzyme bound state. Kinetic studies with the conformationally 'locked' 4,5-O-isopropylidene derivative of 3-dehydroquinic acid and analogs of 5-dehydroquinic acid supports the conformational change hypothesis. The maximum velocity shows no primary deuterium isotope effect indicating that the rate limiting step in the enzyme catalyzed reaction is not proton abstraction as in the analogous non enzymatic reaction. A deuterium isotope effect is observed on the Michaelis constant (K(,m(H))/K(,m(D)) = 0.67 (+OR-) 0.07). This isotope effect may be assigned to proton abstraction which, however, is not rate limiting in the overall enzymatic reaction. Methods have been developed for the synthesis of six substrate analogs having different C-1 substituents (H, CH(,3), Cl, OCH(,3), N(,3) and OH) and the C-5 hydroxyl group inverted in configuration. Studies with these analogs indicate that 3-dehydroquinase is specific for dehydrations only. The absolute configuration of the C-1 position of substrates or competitive inhibitors must be 'R'. The configuration of the C-5 position only affects the affinity of the substrate for the enzyme. Corelation of the thermodynamic 'A' values of the C-1 substituent with the inhibition constants of these analogs, indicates that decreasing 'A' value results in decreasing inhibition constant for substituents not larger than the hydroxyl group. This result is consistent with the conformational change hypothesis. The azido analog was found to be an active site directed photoaffinity label. pH dependence of the maximum velocity and the Michaelis constant suggest the involvement of an ionizing function with a pK of 5.65. This may involve a histidine residue as a general base in the active site of the enzyme