Probing of active site structure of lipoprotein lipase: contribution of activation entropy in the catalysis.

In this study, I have utilized the chromogenic short-chain esters of p-nitrophenol as substrates for probing the active site structure of lipoprotein lipase (LPL). The results indicated that there is a consistent trend in the decrease of the Michaelis-Menten constant with increase of the acyl-chain length. Therefore, it was concluded that the decrease of reactivity with increased chain length is probably not a consequence of a lower affinity of the substrate for the enzyme. The fact that butyrate ester has the optimum acyl-chain length to be a substrate of LPL can be attributed to its chain length being long enough for optimum interaction with the active site His-Ser-Asp triad in forming the transition state complex; yet it is short enough to provide freedom for optimum positioning of the ester bond for transition state complex formation. It is likely that, because of the structural features of the enzyme active site, the increase of the acyl-chain of the substrate from C4 to C5 initiates the contact between the hydrocarbon tail of the acyl-chain and the hydrophobic surface of the active site pocket. Such an interaction, although it causes the stabilization of the ground state enzyme-substrate complex, also causes a large increase in negative activation entropy because of the restricted random motion of the bound substrate. The latter effect is also the likely cause for the progressively lower reactivity of the enzyme with the increase of acyl-chain length above C4, as seen in the LPL-catalyzed lipolysis of monoacid triacylglycerols.