Studies on Enzyme Activation

NIH-supported work in the PI's lab during 2005 - 2008 showed that enzymes utilize the binding energy of their substrate phosphodianions to strongly activate the protein for catalysis of proton transfer (triosephosphate isomerase), hydride transfer (glycerol 3-phosphate dehydrogenase) and decarboxylation (orotidine 5'- monophosphate decarboxylase) reactions. These novel and virtually unprecedented results on protein catalysts, previously studied for more than fifty years, conform to, and affirm, William Jencks' vision about the mechanism by which enzymes achieve their enormous rate accelerations. Substrate phosphodianion activation for three additional enzymes that catalyze reactions of hexose monophosphates in glycolysis or the hexose monophosphate shunt was demonstrated during the previous MIRA. Combining these with our earlier results shows that dianion activation features prominently in the evolution of Nature's most efficient catalytic pathways. It was shown, with MIRA grant support, that the adenosine fragment of adenosine monophosphate (AMP) strongly activates adenylate kinase (AdK) for catalysis of phosphoryl transfer and that the AMP fragment of nicotinamide adenine dinucleotide (NAD) strongly activates three dehydrogenases for catalysis of hydride transfer. Our expanding observations of substrate activation of Nature's most proficient enzyme catalysts spurred the proposal that large substrate binding energies are utilized to drive complex protein conformational changes, transforming flexible inactive enzymes into their catalytically active forms. This proposal challenges protein engineers and computational chemists to pay greater attention to modeling the substrate binding step. MIRA- supported research over the next three years will focus on further expansion of the documented examples of enzymes that undergo substrate-driven activating conformational changes. Substrate activation of three or more of the nine isozymes of human AdK will be characterized to determine if a high nucleoside base specificity for catalysis of phosphoryl transfer to the NMP acceptor is required for effective substrate activation. Several dehydrogenases will be examined to determine if this substrate activation is a conserved and highly propagated mechanism for hydride transfer. We will probe whether the binding energy of the adenosyl moiety of AMP is utilized to activate glucokinase for catalysis of phosphoryl transfer to glucose, in experiments that aim to demonstrate substrate activation for another large class of enzymatic reactions. We will also characterize the architecture of enzymes activated for catalysis by different substrate fragments. One series of experiments will compare the phosphodianion activation sites for enzymes that catalyze reactions of 6-carbon and 3-carbon sugar phosphates. A second campaign will compare the interactions between several dehydrogenases and the activating AMP fragment of the NAD cofactor and examine whether these binding interactions are utilized to create the binding pocket for the hydride donor. A third effort will identify amino acid side chains that participate in adenosyl activation of the phosphoryl transfer reaction catalyzed by human AdK isozyme 1.