DO ENZYMES STABILIZE TRANSITION-STATES BY ELECTROSTATIC INTERACTIONS OR PK(A) BALANCE - THE CASE OF TRIOSE PHOSPHATE ISOMERASE (TIM)

In the triose phosphate isomerase (TIM) catalyzed isomerization of its ligands dihydroxyacetone phosphate (DHAP) and glyceraldehyde phosphate (GAP), it has been established that abstraction of the pro-R hydrogen of DHAP by Glu 165 of TIM initiates the reaction to form an enediolate. However, the question whether a proton is transferred (either in a concerted process or subsequently) to the substrate by the electrophilic His 95 has not been definitively established. We present two sets of calculations that bear on this point: First, we show that intramolecular proton transfer of the hydroxyl hydrogen, the enediolate of DHAP, proceeds with a very small barrier. Second, we show that a model for the enediolate has no intrinsic tendency to accept a proton from an imidazole in the presence of the enzyme environment. This disagrees with the interpretation presented by Bash ct al. (Bash, P. A.; Field, M. J.; Davenport, R. C.; Petsko, G. A.; Ringe, D.; Karplus, M. Biochemistry 1991, 30, 5826) in that they argue for His to donate a proton to the enediolate in the TIM mechanism. Our results could, of course, change upon a more accurate representation of DHAP and the enzyme active site. However, they do suggest that the issue of proton transfer to the (incipient) enediolate is still an open one. In addition, these calculations bear directly on the analysis of Gerlt and Gassman (Gerlt, J. A.; Gassman, P. G. J. Am. Chem. Sec. 1993, 115, 11552) vis-a-vis the putative advantage of an internal pK(a) balance in enzyme active sites.