Stephen J. Benkovic

Professor Benkovic is engaged in a variety of projects connected by the general theme of understanding enzyme catalysis at various levels. On one level is the study of a single enzyme, dihydrofolate reductases, to understand in depth how its structure is harnessed to drive the chemical transformation. On a second level is the study of the eight proteins and enzymes involved in DNA replication by the T4 replisome, to gain a molecular insight as to how these proteins interact within a multi-protein assembly to coordinate leading and lagging strand DNA synthesis. On a third level is the study of de novo purine biosynthesis as it occurs within a human cell line, to determine where in the cell and how the eleven enzymes in the pathway function as a dynamic complex to regulate the metabolic flux. A description of several projects follows.

Structure-function studies on dihydrofolate reductases have revealed the importance of amino acid residues remote from the active site that contribute significantly to the binding of substrate ligands and catalytic turnover. The techniques used include: site specific mutagenesis, pre-steady state kinetics as well as collaborative single molecule kinetics and NMR relaxation measurements. The experimental work is also linked to a strong collaborative theoretical effort. The overall outcome is a collection of evidence that favors a network of residues scattered throughout the enzyme's structural framework that acts in a coupled manner to promote the chemical transformation. This powerful concept may prove to be general and provides a deeper, physical description of what is meant by the concept of "transition state stabilization" that is generally invoked to explain catalysis.

Studies on the T4 DNA replication system provide numerous challenges. How are the eight proteins brought together to constitute the two subassemblies of holoenzyme (a complex of DNA polymerase and an associated clamp protein) and of primosome (a complex of a RNA polymerase, for priming of the lagging strand, and a helicase for unwinding the DNA duplex)? Evidence from fluorescence energy transfer, isocalorimetry, chemical crosslinking, single molecule and ensemble kinetics all point to a stepwise ordered process. Once assembled how is the synthesis of the leading strand and lagging DNA coordinated so both syntheses are completed simultaneously? Answers to these challenging questions are being sought by this powerful combination of biophysical techniques augmented by collaborative crystallographic and electron microscopy studies. To date the data describe a highly orchestrated formation of a replisome consisting of only five of the available proteins with the others acting as catalysts to allow construction to proceed. Once formed the replisome surprisingly is highly dynamic constantly being disassembled and reassembled even during the process of DNA replication.

In vitro investigations of individual enzymes that catalyze the multi-step transformation of a sugar pyrophosphate to a purine have revealed much about their respective mechanism of action. There is little evidence, however, from extra cellular studies for the attractive hypothesis that these enzymes act within a multi-enzyme complex framework. Collaborative efforts using powerful confocal fluorescence methods form the core of an experimental approach to find the locus of these enzymes within eukaryotic cells and to accumulate evidence on their participation in a multi-enzyme complex.

Research on these problems prepares individuals for careers in academics and the biotech and pharmaceutical industries. Many former members of my laboratory have gone on to hold prominent positions in these environments.