Studies on hydrolysis of chiral, achiral and racemic alcohol esters with Pseudomonas cepacia lipase: mechanism of stereospecificity of the enzyme

Abstract

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 K_m values were identical for all the esters, and no nonproductive binding was observed in these substrates. The apparent catalytic constants k_cat 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 L_M 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 L_M of the slow-reacting enantiomer is positioned close to the imidazole ring of the catalytic His and hinders N^ε2 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.