Stephen J. Benkovic was born in Orange, New Jersey near the end of the great depression. He became interested in chemistry at an early age and studied it and English literature at Lehigh University, earning both a B.S. and A.B. degree in 1960. He continued his studies at Cornell University and received a Ph.D. in organic chemistry in 1963. Benkovic then did a 2-year postdoctoral fellowship at the University of California, Santa Barbara with Thomas C. Bruice, with whom he authored what are considered the first bioorganic textbooks focusing on enzyme mechanisms, Bioorganic Mechanisms, Volumes I and II.SJB.gif
In 1965, Benkovic joined the chemistry department at the Pennsylvania State University where he began a lifelong study of enzymes. He was interested in finding out how enzymes worked so quickly and accurately, and he chose bacteriophage T4 DNA polymerase as his model. The prokaryotic enzyme catalyzes the synthesis of DNA and also proofreads and removes nucleotides. Thus it contains both polymerization and hydrolytic activities. Because of these capabilities, in the absence of the next nucleoside triphosphate required to continue polymerization in the 5′ to 3′ direction, the enzyme repeatedly excises and reincorporates the previously incorporated nucleoside. Benkovic took advantage of this "idling" process to determine the stereochemical course of the enzyme's 3′-5′-exonuclease activity in the first Journal of Biological Chemistry (JBC) Classic. He found that the exonuclease proceeds with an inversion of configuration at the phosphorus atom, most probably via a direct displacement by water of the 3′ terminus of the DNA chain to yield a 5′-nucleotide. This was the first example of phosphodiester hydrolysis catalyzed by an exonuclease that did not involve a covalent phosphoryl-enzyme intermediate.
In the second Classic, Benkovic looks at the spatial relationship between the polymerase and exonuclease active sites. Using a bulky biotin-streptavidin block at a specified position in an oligonucleotide sequence, he was able to monitor the closest distance of approach of the T4 enzyme before being prevented from performing either of its activities by the biotin- streptavidin complex. His results indicated a distance of 4-5 nucleotides between the biotin-streptavidin probe and the exonuclease site and a possible separation of 2 nucleotides between the exonuclease and polymerase active sites.
Benkovic has continued to study DNA replication and is now looking at the assembly and function of the T4 replisome. He is currently the Evan Pugh Professor of Chemistry and holds the Eberly Chair in Chemistry at Penn State.
His work has been recognized by awards and fellowships including: Alfred P. Sloan Fellowship, NIH Career Development Award, Guggenheim Fellowship, the Pfizer Award in Enzyme Chemistry, the Gowland Hopkins Award, the Repligen Award for Chemistry of Biological Processes, the Alfred Bader Award, the Chemical Pioneer Award from the American Institute of Chemists, and the Christian B. Afinsen Award, the Nakanishi Prize, the Benjamin Franklin Medal in Life Science, the Ralph F. Hirschmann Award in Peptide Chemistry, the 2009 National Medal of Science and the National Academy of Science Award in Chemical Sciences in 2011. In addition, he has been elected to memberships in the American Academy of Arts and Sciences, the Institute of Medicine, National Academy of Sciences, and the American Philosophical Society. A complete list of honors and awards are listed below.
2011- National Academy of Sciences Award in Chemical Sciences
2010- National Medal of Science, National Medals of Science and Technology and Innovation
2010- Ralph F. Hirschmann Award in Peptide Chemistry
2009- Benjamin Franklin Medal in Life Science
2006- Royal Society Centenary Lecturer
2005- Nakanishi Prize (ACS)
2002- American Philosophical Society
2000- Christian B. Anfinsen Award
1995- Honorary Doctorate of Science (Lehigh University)
1995- Alfred Bader Award
1994- Member of the Institute of Medicine, National Academy of Sciences
1989- The Repligen Award
1988- Arthur C. Cope Scholar Award
1986- The Eberly Chair in Chemistry
1986- Gowland Hopkins Award
1985- Member of the National Academy of Sciences
1984- Fellow of the American Academy of Arts and Sciences
1977- Evan Pugh Professorship
1977- Pfizer Enzyme Award
Benkovic's recent work has focused on the assembly and kinetic characteristics of the protein machinery that is responsible for DNA replication by T4 phage and yeast; the importance of dynamic coupling of proximal and distal residues in the catalytic cycle of the dihydrofolate reductase enzyme that serves as a paradigm for describing enzymic catalysis in terms of a series of orchestrated protein conformations; the direct intracellular observation by fluorescent imaging of de novo purine biosynthesis, and the development of novel cyclic peptides for modulating protein/protein interactions.
A DNA polymerase is an enzyme which helps in catalyzing the polymerization of deoxyribonucleotides into a DNA strand. Benkovic's work in unraveling the intricate functioning of DNA polymerases led to his being awarded the Repligen award in 1989. Most of the research in this field before Benkovic's time had strayed away from true facts simply because it was impossible at that time to watch proteins and replication systems at work in vivo. But Benkovic, in his time brought together tools from different fields of science so as to create a mechanistic understanding of how these systems worked and that is why he is considered to be among the most prominent mechanistic enzymologists in the world.
Early research had identified the key enzymes responsible DNA replication. However, most of this research had focused on the end results of the replication process. Benkovic took off from there and studied the precise mechanisms of these enzymes by following up on each step of the process. The research he undertook was on multi-enzyme systems, specifically the DNA replication in T4 phages, a virus which infects E.Coli. His work disproved many of the long held false assumptions on DNA replication in addition to explaining the process in detail and revealing many of the additional roles of these enzymes.
One of his earliest works1 describes a possible mechanism for the 3' 5' exonuclease activity of T4 DNA polymerase when operative under idling conditions. This enzyme, which possesses both polymerization and hydrolytic capabilities, was used along with (SP)-2'-deoxyadenosine-5'-O-(1-thio (1-18O2) triphosphate) and poly (d (A-T)). Poly (d (A-T)) template primer so as to understand the stereochemical course of the enzyme's exonuclease activity. Absence of one of the complementary nucleoside triphosphates during DNA replication led to the establishment of an idling process by which one or more of the available complementary nucleoside triphosphates were continuously incorporated and subsequently excised. Since it had been earlier established that the process of polymerization results in an inversion in configuration at Pα2, the absence of any change in configuration at Pα after both polymerization and excision had taken place led to the conclusion that the exonuclease activity of the enzyme can be best explained by a direct displacement of the 3' terminus of the DNA chain by water to yield a 5' nucleotide. Interestingly, in one of his papers3 published two years later, it was established that the 3' 5' exonuclease activity of E. Coli DNA Polymerase I can be explained by the same mechanism mentioned above.
The E. Coli DNA Polymerase I is a multifunctional enzyme responsible for the repair and replication of DNA in vivo. Besides its polymerase activity, this enzyme is also capable of 3' 5'and 5' 3' exonucleolytic action. The enzyme (103 kDa) is capable of undergoing limited proteolysis to give two fragments: a large fragment; Klenow (68 kDa) which retains the polymerase and the 3' 5'exonuclease activities and a smaller fragment (36 kDa) which retains the 5' 3' exonuclease activity5.
Benkovic's study on the polymerization reaction catalyzed by E. Coli DNA Polymerase I using poly (dA).oligo (dT) homopolymer template- primer system led to the revelation that the reaction follows an ordered mechanism in which the enzyme first combines with the template-primer to form an E.Poly complex followed by the addition of MgTTP and catalysis. The steady-state isotope-partitioning experiments suggested the presence of two partially rate-determining steps, one of which precedes the actual chemical phosphodiester bond-forming step (k=4.6 s-1) and the other which follows this step (k=4.0 s-1). Binding of MgTTP to E.Poly complex was shown to be a rapid equilibrium step as well. The minimal reaction scheme for the polymerization reaction catalyzed by DNA polymerase I is shown below.
It was proposed in the paper that the first rate determining step is the one which follows TTP binding and precedes the chemical bond forming step. Since it is believed that the specificity of the DNA polymerase enzyme depends on the base pairing between the template and the nucleotide being introduced, it was inferred that when the correctly base paired nucleotide triphosphate is in the active site, the enzyme may respond by changing its conformation, so that the following catalytic steps can occur.
Although it was difficult to assign the second rate determining step to a particular physical event using the data from the study conducted, it was proposed that the second step might in fact, correspond to the translocation of the newly elongated primer out of the TTP binding site, possibly by another conformational change in the enzyme, so as to return the E.Poly complex to the initial open configuration with an open binding site for the next TTP to bind.
Since the paper mentioned above pointed at the existence of two partially rate determining steps, one of the questions which followed it was whether there were any covalent nucleotidyl-enzyme intermediates along the associated pathways. A paper by the same author6 published in 1985 disproved their existence.
The very next year, Benkovic and his co-workers published a paper7 explaining a possible mechanism of the idling turnover reaction catalyzed by the Klenow fragment of E. Coli DNA Polymerase I. The results of the mechanistic studies conducted in this paper revealed that the 3'-terminal deoxynucleotide residue of the DNA substrate forms two products in the reaction, the 5'-monophosphate and the 5'-triphosphate products. While the dNMP was accounted for by the 3' 5' exonucleolytic activity of the enzyme, the appearance of dNTP could not be explained right away. Finally, it was surmised that since each of the polymerization events releases an equivalent of PPi, the dNTP pool may provide the source of PPi required for this kind of cleavage and the following scheme was proposed.
A paper by the same authors8 published in the same year attempted to explain the exonuclease to polymerase activity switch involved in the idling turnover reactions. According to the study, it was concluded that the reaction involves a 3' 5' exonucleolytic removal of the terminal residue followed by a rapid polymerization step so as to insert the correct nucleotide and that further exonuclease action of the enzyme is suppressed by the conversion of the enzyme from the exonuclease to a polymerase mode when a complementary dNTP pool is made available. The next question to be addressed was regarding the relative positions of the exonuclease and polymerase active sites on the enzyme with respect to each other and as to whether it was possible for the enzyme to switch between the two modes of action within a single binding event to its DNA substrate. Although the location of the active sites could not be confirmed by the studies, the results of the isotope-trapping experiments suggested that the DNA within the enzyme-DNA complex is capable of equilibrating between the active sites, if they are indeed separate.
Interestingly, the same author published a paper three years later11 showed that the polymerase and exonucleolytic active sites of the Klenow fragment are separate and have different structural requirements for the DNA to act as a substrate. It was seen that while the polymerase site of the Klenow fragment does not require that the DNA strands be separated for it to act as a substrate, the exonucleolytic site does require that the primer terminus be at least four bases from the nearest cross-linked base pair. The results of this study were found to be consistent with the observation of Joyce and Steitz (1987) who concluded from the X-Ray crystallographic data of Klenow fragment that the polymerase and exonucleolytic active sites are about 30 AO apart from each other. It was thus concluded that the separation between the active sites observed in the crystallized enzyme is preserved in the functional enzyme in solution.
Another study9 employed isotope-trapping and rapid kinetic techniques to study the individual steps involved in Klenow catalyzed single nucleotide incorporation and pyrophosphorolysis reactions using a synthetic DNA substrate of defined sequence. The study concluded that a step other than the chemical bond formation step is the rate limiting step and proposed a kinetic scheme for the Polymerase I catalyzed polymerization which accurately predicted both the polymerization as well as the pyrophosphorolysis reactions. According to the scheme, the rate determining step is a conformational change preceding catalysis. It could not however, explain the role of the conformational change.
Benkovic and co-workers (1988)10 used short DNA oligomers of defined sequence to explain the mechanism by which the Klenow fragment of DNA Polymerase I polymerizes the correct and incorrect deoxynucleotide triphosphates. It was proposed by them that the high fidelity of the polymerization reaction catalyzed by DNA Polymerase I is because of a three stage mechanism for discriminating errors. The first step of discrimination prevents most of the misincorporation and is constituted by the high free energy barrier of the chemical step along with a moderate discrimination of the nucleotide triphosphates which bind to the DNA substrate during the phosphodiester bond formation. The second step is a step of no obvious chemistry in which the enzyme undergoes a conformational change, thus allowing the rather inefficient exonuclease to remove most of the mismatches. The third step, a step of classical proofreading slowly adds the next correct dNTP onto a mismatch, allowing the proofreading exonucleases to remove the mismatch. The study could not, however, explain the precise physical or chemical nature of these three steps.
A study on the inactivation of DNA Polymerase I (Klenow fragment) by adenosine 2', 3'- epoxide 5'- triphosphate12 showed that epoxy-ATP is capable of suppressing the polymerase activity of Klenow fragment and is resistant to degradation by the 3' 5' exonuclease activity of the enzyme. However, epoxy-ATP was found to be incapable of suppressing the exonuclease activity of the enzyme along with its polymerase activity and this fact led to the possibily that even though the polymerase active site of the Klenow fragment be occupied with a duplex DNA, the enzyme might still be able to simultaneously bind to a second DNA duplex and carry out an exonucleolytic cleavage.
In another study13, short duplex DNAs fluorescently labeled at a specific base were constructed by annealing fluorescent derivatives of short oligonucleotides of defined sequence to their complementary oligomers and were used to study the points of strong enzyme-DNA interactions by varying the positions of the label within the duplex DNA. The idea behind this study was that the fluorescent emission from a labeled DNA duplex would increase on binding to the Klenow fragment of the DNA Polymerase I. The labeled DNA duplexes were found to be highly efficient in this study.
Resonance energy transfer experiments were used to determine the separation distances between fluorescent derivatives of substrates for the Klenow fragment and a unique sulfhydryl cysteine 907 on the enzyme. Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates and deoxynucleotide monophosphates were used in this study.
