In 1965, he became a member of the Chemistry Department atPenn State University, and later in 1970, he was promoted to the position of full professor of chemistry.[2] He received further recognition in 1977 as an Evan Pugh Professor of Chemistry and in 1988 as the holder of the Eberly Chair in Chemistry.[1]
Benkovic has made contributions that have impacted our understanding ofbiological processes. He was among the first to hypothesize that conformational changes outside an enzyme's active site were necessary for achieving maximalcatalysis.[6] This was illustrated in his studies ondihydrofolate reductase (DHFR) that identified dynamic structural changes and their time scale that optimized the enzyme turnover.[7][8] He showed how multi-enzyme complexes are assembled to achieve specificity and function and where several activities are present how they are integrated.[9] This was accomplished in his studies onDNA replication that featured the assembly, disassembly and function of the T4 replisome that coordinates DNA replication.[10] Benkovic discovered the first example of a reversible metabolon, thepurinosome in de novo purine biosynthesis, that only assembles in response to cellular demands and acts temporally and spatially to deliver neededmetabolites to cellular constituents.[11][12]
Conformational Movements
A major theme of Benkovic's research has been understanding the source of the efficiency of enzymatic catalysis.[13][14] He first dissected into individual steps the catalytic cycle used bydihydrofolate reductase (DHFR) using pre-steady-state methods and then tied the contribution of variousamino acids, both within and outside the active site, to specific steps.[8][15] Significant changes in the rates of hydride transfer were not limited to active-site residues, nor were the effects of multiple mutations additive in terms of free energy. The amide backbone and side chains of these distal residues were found by NMR to be in regions of high frequency motion (n-psec) and by molecular dynamic simulations the motions of these distal residues were found to be coupled.[16] Genomic analysis of multiple DHFR sequences revealed low overallDNA sequence homology (30%), but surprisingly high conservation in the same regions whoseamino acids had been implicated in catalysis by kinetic analysis, NMR measurements, and molecular dynamics simulations.[6] The latter directly incorporated these distal residues into a network that acted along the reaction coordinate to facilitate the hydride transfer.[13][17]
Illustration
This concept was further elaborated to posit that the measured rates of steps that constitute the turnover cycle of DHFR represent the rates of the conformational changes required to execute the chemical transformation.[6] The enzymic reaction is not limited by the energetics of the chemical reaction but by the mechanics of sampling that occur within the enzyme substrate complex.[18]
This concept of biological catalysis has the enzyme's highly pre-organizedMichaelis complex with its active-site residues and substrates juxtaposed by using the dynamics of the protein fold to sample substrate and active site conformations in order to find those optimal for the chemical transformation. The actual chemistry of bond breaking and forming is fast relative to the sampling process. Only a small change triggered by movement within the protein fold along a network of coupled residues is needed to surmount the reaction barrier.[13] The protein fold dictates the type of chemistry that a class of enzymes can accomplish (a rationale for the common mechanistic element extent in protein super-families); allosteric effects are a consequence of creating or inhibiting such networks and drugs can be designed that target such networks.[19][20] It also explains the generally low catalytic activity of more rigid structures such asmacrocycles andantibodies.[21]
A multi-enzyme complex for the replication of DNA—the T4 replisome
Of particular importance is how multiple protein systems such as thereplisomes responsible forDNA replication function whereprotein-protein interactions create a large catalytic network. The T4 replisome can be assembled in vitro from eight separate proteins into the four units that catalyze leading and lagging strand synthesis at a replication fork.[10] With a functioning replisome capable of leading/lagging strand synthesis in hand, key discoveries of broad interest applicable to other replisomes were made. Firstly thepolymerase actively exchanges in/out of the twoholoenzymes within thereplisome thus providing a "remodeling" flexibility for the repair of stalled replication forks that occur on damaged DNA strands by other lesion bypasspolymerases.[22] Secondly, two mechanisms dictateOkazaki fragment length: the classical collision mechanism where a finished Okazaki fragment abuts the previous one releasing the lagging strand polymerase and the signaling mechanism where the lagging strand polymerase recycles before the completion of the previous Okazaki fragment.[10] This feature is essential to maintain coordinated leading/lagging strand synthesis.[23]
FGMAS GART
De Novo Purine Biosynthesis by a Purinosome Metabolon
De Novo Purine Biosynthesis
A longstanding question incellular metabolism is how metabolic enzymes in a given network organize within thecytosol, densely packed with myriad proteins andmetabolites, to facilitatemetabolic flux. One solution is through the formation of amacromolecular complex of enzymes, termed a 'metabolon'.[24] Thede novo purine biosynthetic pathway is a highly conserved, energy-intensive pathway that generates inosine 5ᶦ-monophosphate (IMP) fromphosphoribosylpyrophosphate (PRPP).[25] In humans, this metabolic transformation is carried out in ten steps by the sequential orchestration of the activities of the six enzymes.[26] Evidence that the enzymes might condense within cells to form the purinosome derived from confocal microscopy onHeLa cells using chimeric constructs of these enzymes that revealed in common merged punctates for the six enzymes as illustrated for the two enzymes, FGAMS and GART.[27][28] Unlike more traditional staticmetabolons,purinosome formation is a reversible process.[24] Spatial control of purinosome assembly in HeLa cells was found to bemicrotubule assisted and to colocalize withmitochondria as shown by super resolution chemical imaging.[29] De novo purine biosynthesis is likely most efficient whenpurinosomes are located nearmitochondria to capture needed substrates exported from the mitochondria.[30]
Fierke, C. A., Johnson, K. A., and Benkovic, S. J. (1987) Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase fromEscherichia coli,Biochemistry26, 4085-4092.[8]
Rajagopalan, P. T. R., Lutz, S., and Benkovic, S. J. (2002) Coupling interactions of distal residues enhance dihydrofolate reductase catalysis: Mutational effects on hydride transfer rates,Biochemistry41, 12618-12628.[15]
Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Dynamics of the dihydrofolate reductase—folate complex: Catalytic sites and regions known to undergo conformational change exhibit diverse dynamical features,Biochemistry34, 11037-11048.[16]
Benkovic, S. J. and Hammes-Schiffer, S. (2003) A perspective on enzyme catalysis,Science301, 1196-1202.[13]
Agarwal, P. K., Billeter, S. R., Rajagopalan, P. T. R., Benkovic, S. J., and Hammes-Schiffer, S. (2002) Network of coupled promoting motions in enzyme catalysis,Proc. Natl. Acad. Sci. USA99, 2794-2799.[17]
Hammes-Schiffer, S. and Benkovic, S. J. (2006) Relating protein motion to catalysis,Annu. Rev. Biochem.75, 519-541.[6]
Lee, J., Natarajan, M., Nashine, V. C., Socolich, M., Vo, T., Russ, W. P., Benkovic, S. J., and Ranganathan, R. (2008) Surface Sites for Engineering Allosteric Control in Proteins, Science 322, 438-442.[20]
Goodey, N. M. and Benkovic, S. J. (2008) Allosteric regulation and catalysis emerge via a common route, Nat. Chem. Biol. 4, 474-482.[19]
Lerner, R. A., Benkovic, S. J., and Schultz, P. G., (1991) At the Crossroads of Chemistry and Immunology: Catalytic Antibodies, Science 252, 659 667.[21]
Yang, J., Zhuang, Z., Roccasecca, R. M., Trakselis, M. A., and Benkovic, S. J. (2004) The dynamic processivity of the T4 DNA polymerase during replication,Proc Natl. Acad. Sci. USA101, 8289-8294.[22]
Benkovic, S.J., Spiering, M.M. (2017) "Understanding DNA Replication by the Bacteriophage T4 Replisome", JBC, 292 (45) 18434-18442.[10]
An, S., Kumar, R., Sheets, E. D., and Benkovic, S. J. (2008) Reversible compartmentalization of de novo purine biosynthetic complexes in living cells, Science 320, 103-106.[27]
French, J. B., Jones, S.A., Deng, H., Hu, H., Pugh, R. J., Chan C. Y., Kim, D., Pedley, A. M., Zhao, H., Zhang, Y., Huang, T. J., Fang, Y., Zhuang, X., and Benkovic, S. J., (2016) Spatial colocalization and functional link of purinosomes with mitochondria, Science, 351:6274, 733-736.[28]
Pedley, A.M., Pareek, V., Benkovic, S.J. (2022) The Purinosome: A Case Study for a Mammalian Metabolon, Annu. Rev. of Biochem., Volume 91:89-106.[30]
Rock, F. L., Mao, W., Yaremchuk, A., Tukalo, M., Crepin, T., Zhou, H., Zhang, Y.-K., Hernandez, V., Akama, T., Baker, S. J., Plattner, J. J., Shapiro, L., Martinis, S. A., Benkovic, S. J., Cusack, S., and Alley, M. R. K. (2007) An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site,Science 316, 1759-1761.[31]
^Cameron, C. E.; Benkovic, S. J. (1997-12-16). "Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant".Biochemistry.36 (50):15792–15800.doi:10.1021/bi9716231.ISSN0006-2960.PMID9398309.
^abcFierke, C. A.; Johnson, K. A.; Benkovic, S. J. (1987-06-30). "Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli".Biochemistry.26 (13):4085–4092.doi:10.1021/bi00387a052.ISSN0006-2960.PMID3307916.
^abRajagopalan, P. T. Ravi; Lutz, Stefan; Benkovic, Stephen J. (2002-10-22). "Coupling interactions of distal residues enhance dihydrofolate reductase catalysis: mutational effects on hydride transfer rates".Biochemistry.41 (42):12618–12628.doi:10.1021/bi026369d.ISSN0006-2960.PMID12379104.
^abEpstein, D. M.; Benkovic, S. J.; Wright, P. E. (1995-09-05). "Dynamics of the dihydrofolate reductase-folate complex: catalytic sites and regions known to undergo conformational change exhibit diverse dynamical features".Biochemistry.34 (35):11037–11048.doi:10.1021/bi00035a009.ISSN0006-2960.PMID7669761.
^abGoodey, Nina M.; Benkovic, Stephen J. (2008-08-01). "Allosteric regulation and catalysis emerge via a common route".Nature Chemical Biology.4 (8):474–482.doi:10.1038/nchembio.98.ISSN1552-4469.PMID18641628.
