EFFECTS OF CHANGING GLUTAMATE-487 TO LYSINE IN RAT AND HUMAN LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE - A MODEL TO STUDY HUMAN (ORIENTAL TYPE) CLASS-2 ALDEHYDE DEHYDROGENASE

Many Oriental people possess a liver mitochondrial aldehyde dehydrogen ase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase act ivity. To investigate the cause of the lack of activity of this aldehy de dehydrogenase, we mutated residue 487 of rat and human liver mitoch ondrial aldehyde dehydrogenase to a lysine and expressed the mutant an d native enzyme forms in Escherichia coli. Both rat and human recombin ant aldehyde dehydrogenases showed the same molecular and kinetic prop erties as the enzyme isolated from liver mitochondria. The E487K mutan ts were found to be active but possessed altered kinetic properties wh en compared to the glutamate enzyme. The K-m for NAD(+) at pH 7.4 incr eased more than 150-fold, whereas k(cat) decreased 2-10-fold with resp ect to the recombinant native enzymes. Detailed steady-state kinetic a nalysis showed that the binding of NAD(+) to the mutant enzyme was imp aired, and it could be calculated that this resulted in a decreased nu cleophilicity of the active site cysteine residue. The rate-limiting s tep for the rat E487K mutant was also different from that of the recom binant rat liver aldehyde dehydrogenase in that no pre-steady state bu rst of NADH formation was found with the mutant enzyme. Both the rat n ative enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophil ic attack. Each enzyme form showed a 2-fold activation upon the additi on of Mg2+ ions. Substituting a glutamine for the glutamate did not gr ossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD(+) for the K-i of NADH was the same in the lysine enzyme as it was in the glutamate form . Because of the altered NAD(+) binding properties and k(cat) of the E 487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.