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Chemistry

Research Interests

Enzyme and Organic Reaction Mechanism

We have been studying the reaction catalyzed by the enzyme ketol acid reductoisomerase. This enzyme functions in the biosynthetic pathway of the branched chain amino acids. We determined the kinetic mechanism of the enzyme and studied the chemical mechanism by using a combination of synthetic alternative substrates, kinetic isotope effect studies and the pH dependence of the kinetic parameters for the enzyme reaction. Using DNA cloning, sequencing and site-directed mutagenesis techniques, we were able to identify the specific function that several amino acids have in the catalysis of the reaction. We were able to identify the enzyme region responsible for the binding of the co-substrate NADPH by creating a series of site specific mutations in the enzyme. This mutant enzyme was then studied to determine if the amino acid changes affected the rate of inhibition induced by a slow-tight binding inhibitor. We plan to continue our studies by using stopped-flow kinetic studies to measure the rates of specific steps in the chemical mechanism.

We also are studying the mechanisms of several enzymes in the NAD biosynthetic pathway. We have examined quinolinate phosphoribosyl transferase and plan to clone two other enzymes of this pathway. In particular, we plan to use PCR to clone the genes for aspartate oxidase and quinolinate synthase. Once these genes are cloned, we will use molecular biology techniques to over-express the enzymes. Once this is accomplished, we will initiate a mechanistic study of the enzyme catalyzed reactions.

In collaboration with Dr. Gerald Koser, we are synthesizing a series of nucleoside analog bis-ketol phosphate triesters. These compounds, by virtue of the triester functionality, are able to deliver nucleotide analog monophosphates to cells. These nucleotide analogs then function as antiviral or anticancer drugs by inhibiting either reverse transcriptase or DNA polymerase.

 Calvo Figure 1

Figure 1. HPLC-MS analysis of tryptic peptides from QPRTase. a) Native QPRTase was subject to trypsin digestion. The peptides were separated on a reverse phase VYDAC 218TP54 column interfaced to the electrospray mass spectrometer. The vertical axis as A210 b) TNBS modified QPRTase was digested with trypsin and the peptides separated. The vertical axis is A345. At 345 nm only trinitrophenyl modified peptides are detected.

Selected Publications

  1. Rajula Bhatia Gau, Tanesha Roberts, and K.C. Calvo. Lysine 70 of E. coli Quinolinate Phosphoribosyltransferase is Protected from Chemical Modification by Formation of an Inhibitor Complex, Protein and Peptide Letters, 13, 163-167, 2006.
  2. "Synthesis and Anti-HIV-1 Activity of Bis-ketol AZT Monophosphates," Koser, G.; Huang, Y.; Chen, K.; and Calvo, K., J. Chem. Soc. Perkins Trans. 1, 1995, 299-302.
  3. "Cloning, Sequencing, Purification and Characterization of Quinolinate Phosphoribosyl Transferase from E. Coli," Bhatia, R., and Calvo, K., Arch. Biochem. Biophys. 1996, 325(2), 270-78.
  4. "Bis-Ketol Phosphate Alkyl Triesters: Rate of Initial Ketol Group Hydrolysis," Calvo, K.; Moore, R.; and Koser, G., Tetrahedron Letters, 1996, 37(8), 1169-72.

Calvo Photo

KIM C. CALVO

Associate Dean, Arts & Science
Professor

B.A., 1973, The Ohio State University
Ph.D., 1981, The Ohio State University
Postdoctoral fellow, 1981-1984, Harvard University

Office: KNCL 207
(330) 972-6078

Lab: KNCL 215

Email: kcalvo@uakron.edu

Website:

The University of Akron

Akron, OH 44325
Phone: 330-972-7111
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