Making an Oriental equivalent of the yeast cytosolic aldehyde dehydrogenase as well as making one with positive cooperativity in coenzyme binding by mutations of glutamate 492 and arginine 480

Yeast has at least three partially characterized aldehyde dehydrogenases. P revious studies by gene disrupted in our laboratory revealed that the Sacch aromyces cerevisiae cytosol ALDH1 played an important rare in ethanol metab olism as did the class 2 mitochondrial enzyme. To date, few mutagenesis stu dies have been performed with the yeast enzymes. An important human variant of ALDH is one found in Asian People. In it, the glutamate at position 487 is replaced by a lysine. This glutamate interacts with an arginine (475) t hat is located in the subunit that makes up the dimer pair in the tetrameri c enzyme. Sequence alignment shows that these two residues are located at;p ositions 492 and 480. respectively, in the yeast class 1 enzyme which share s just 45% sequence identity with the human enzymes. Mutating glutamate 492 to lysine produced an enzyme with altered kinetic properties when compared to the wild-type glutamate-enzyme. The K-m for NADP of E492K increased to nearly 3600 muM compare to 40 muM for wild-type enzyme. The specific activi ty decreased more than IO-fold with respect to the recombinant wild-type ye ast enzyme. Moreover. substituting a glutamine for a glutamate was not detr imental in that the E492Q had wild-type-like K-m for NADP and V-max. These properties were similar to the changes found with the human class 2 E487K m utant form. Further, mutating arginine 480 to glutamine produced an enzyme that exhibited positive cooperativity in NADP binding. The K-m for NADP inc reased 11-fold with a Hill coefficient of 1.6. The NADP-dependent activity of R480Q mutant was 60% of wild-type enzyme. Again, these results are very similar to what we recently showed to occur with the human enzyme [Biochemi stry 39 (2000) 5295-5302]. These findings show that the even though the glu tamate and arginine residues are not conserved, similar changes occur in bo th the human and the yeast enzyme when either is mutated. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.