PROTON DIFFUSION IN THE ACTIVE-SITE OF TRIOSEPHOSPHATE ISOMERASE

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is∼104 s−1. T-Labeled substrate is found to lose ~95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E•enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated. The evidence supporting internal exchange is that T, which is exchanged into triose-P isomerase by brief exposure to TOH, is found in substrate after the enzyme is diluted into a large volume of unlabeled water containing substrate. The evidence for external exchange is that loss of T introduced in this way is competitive with substrate concentration. It is suggested that a major factor in the evolution of some enzymes is the need to keep the intermediate state and its associated amino acids isolated from the medium to prevent the loss of functional protons. The extent to which DHAP becomes labeled in a chase is not greatly altered if phosphoglycolate is also present at concentrations that inhibit the steady-state rate ~90%. The T present on the E•inhibitor complex must be protected from exchange with the medium so that it will be trapped by DHAP after the inhibitor dissociates. © 1990, American Chemical Society. All rights reserved.