STUDIES ON HYDROLYSIS OF CHIRAL, ACHIRAL AND RACEMIC ALCOHOL ESTERS WITH PSEUDOMONAS CEPACIA LIPASE : MECHANISM OF STEREOSPECIFICITY OF THE ENZYME

Steady-state kinetics of Pseudomonas cepacia lipase-catalysed hydrolysis of five analogous chiral and achiral substrates, i.e. (R)- and (S)-1-methyl-2-(4-phenoxyphenoxy)ethyl acetates (R)- and (S)-1a, (R)- and (S)-2-methyl-2-(4-phenoxyphenoxy)ethyl acetates (R)- and (S)-1b and 2-(4-phenoxyphenoxy)ethyl acetate 1c, were investigated in sufficiently emulsified reaction mixtures of water-insoluble substrates. The apparent Michaelis constant Km values were identical for all the esters, and no nonproductive binding was observed in these substrates. The apparent catalytic constants kcat were found to reflect the leaving abilities of the alcoholate ions for the fast-reacting enantiomers. These observations, based on the findings that acyl-enzyme intermediate formation was rate-determining in the overall reaction, strongly suggested that all the substrates are bound to the enzyme in the same manner whether or not the alcohol moiety has a medium-sized substituent LM at the stereocentre and that the breakdown of a tetrahedral intermediate is rate-determining in the acylation of the enzyme. Time courses were also studied for the hydrolysis of racemic 1-ethyl-2-(4-phenoxyphenoxy)ethyl acetate 1d together with 1a, 1b and 1c. The enzyme distinguished (R)-1d from its antipode perfectly and hydrolysed only the (R)-enantiomer. These results were interpreted to indicate that LM of the slow-reacting enantiomer is positioned close to the imidazole ring of the catalytic His and hinders NIµ2 of the residue from forming a weak interaction with O1 of the leaving alcohol and that the breakdown of the tetrahedral intermediate is thus inhibited.