Dept. of Biochemistry & Molecular Biology
Oregon Health & Science University
Phosphoryl transfer is ubiquitous in biology. Defects in the enzymes that catalyze such transfer upset normal cellular energy metabolism, motility and the generation of transmembrane potentials. In “high energy” phosphoanhydride bonds, such as those in ATP and GTP, computational evidence has emerged showing a dependence of phosphoanhydride bond energy and reactivity on the anomeric effect. The hypothesis guiding this dissertation research is that enzymes have evolved to take advantage of an intrinsic anomeric effect in nucleotide ligands to drive catalysis. The first section addresses how enzymes might modulate the anomeric effect for catalytic gain. The potential impact of hydrogen bonding from neighboring molecules is calculated using high level quantum mechanical analysis. It is found that hydrogen bonds between water and the terminal phosphoryl group of an ATP analog induce a reduction in the anomeric effect and a shortening/strengthening of the scissile phosphoanhydride bond. Although the anomeric effect requires strict orbital alignments, geometrical requirements for hydrogen bonds are minimal. Thus a larger array of interactions than previously thought has the potential to modulate the strength of the scissile bond. The second part of the dissertation addresses whether enzymes have evolved the potential to modulate bond strength through the anomeric effect. A top-down analysis of protein Data Bank structures was performed to examine active site interactions that might be prevalent in phosphoryl transfer enzymes, the hypothesis being that these enzymes might have interactions that would optimize hyperconjugation. The database survey highlighted several key interaction patterns. Particularly abundant in phosphoryl transfer x enzymes, were hydrogen bonds with the bridging oxygen of the scissile phosphoanhydride bond. Computation shows these interactions elongate the scissile bond by enhancing the anomeric effect. Experimental mutagenesis against highlighted interactions in arginine kinase resulted in a 100-fold decrease in catalytic rate, providing both evidence for a relationship between the anomeric effect and catalytic rate and support for the theory that the anomeric effect is a factor in AK catalysis. There is increasing appreciation that enzymes can employ multiple mechanisms of rate enhancement. This dissertation research uncovered a hitherto overlooked mechanism to accelerate phosphoryl transfer that has not yet proven to be the dominant means of rate enhancement in any enzyme, but appears to be an important component in many enzymes, establishing a common denominator between families of enzymes that were previously thought to operate with quite distinct mechanisms of action.
School of Medicine
Summerton, Jean C., "The Anomeric Effect in Enzyme-catalyzed Phosphoryl Transfer" (2014). Scholar Archive. 3483.