Are Enzyme Active Sites Built in Multiple Layers?

Northeastern University, Boston MA

Investigators

Abstract

The objective of this project is to obtain greater insight into how nature constructs enzyme active sites. The project aims to establish the principle of multilayer enzyme active sites and to gain understanding of how residues outside the first layer influence the catalytic activity and specificity. This understanding will have important implications for enzymology and for protein engineering. Experiments and calculations are designed to understand how the layers beyond the first one contribute to catalysis. In recent decades, structural and biochemical studies have identified residues in active sites of enzymes that directly participate in substrate binding and/or in the chemical transformation steps. These residues generally are in direct contact with the reacting substrate molecule and may be thought of as located in a "first shell" surrounding the reacting species. This project builds upon preliminary evidence that residues beyond the first shell also are important for catalytic function. The focus here is primarily on the second layer, the residues in contact with the first-shell residues. Systematic experimental studies to establish the importance of second-shell residues in enzyme catalysis, and computational studies to understand the varied roles that they play in enzymatic function will be pursued. In this project, site-directed mutagenesis experiments and kinetics assays will be performed on selected enzymes. These proteins will be chosen to represent different kinds of enzymes with different degrees of predicted participation by residues outside the first shell. Crystal structures of the mutants will be determined to test for structural changes upon mutation. Electrostatics calculations and molecular dynamics simulations will be performed to understand the mechanisms by which second-shell residues participate in function. A better understanding of how enzymes affect catalysis can help to produce cleaner, lower cost, "green" industrial processes and to the commercially viable enzymatic synthesis of biofuels. Students will be trained in computational methods, in protein expression, mutation, purification, kinetics assays, and crystal structure determination. Part of the project will be integrated into the undergraduate Chemical Biology laboratory course. Ongoing outreach efforts to minority students, particularly Native Americans, include LSAMP student participation in this research project and a hands-on computer lab demonstration to middle school students. A tracking system will be put into place to follow the careers of research group alumni and will attempt to quantify the impact of undergraduate research upon the future careers of bachelor's degree students.