Controlling protein evolution.

A method is presented for selecting, with a high degree of specificity, mutants for the enzyme alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae having altered kinetics. The method depends on the facts that a) petite yeast strains (those unable to respire aerobically) have an absolute dependence on the presence of cytoplasmic alcohol dehydrogenase, and b) allyl alcohol is readily oxidized by yeast ADH to the highly poisonous product acrolein. If petite yeast are grown in the presence of allyl alcohol surviving mutants show alterations that can be traced to the ADH structural gene. Eight out of nine spontaneous mutant enzymes and five out of five mutants enzymes induced by nitrosoguanidine exhibited a slower electrophoresis mobility than wild type. There may therefore be some correlation between electrophoretic mobility and function. Of three mutant enzymes purified, one of spontaneous origin and two induced by nitrosoguanidine, each showed a different pattern of altered kinetics and a different specific activity. This strongly suggests that, despite the considerable specificity of the selective procedure, there are many ways in which an enzyme can be altered in response to an environmental stimulus. The implications of these results for the study of protein evolution and some directions for future research are briefly discussed.