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.
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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.
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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.
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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.
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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.
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