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Enzyme Mechanisms

Professor Kuchta's group is primarily interested in the broad area of mechanistic enzymology. Their studies are directed towards understanding enzymic catalysis at the level of individual steps in the reaction pathway. To accomplish this, a variety of steady-state and pre-steady-state kinetic techniques, protein modification reagents, and inhibitors are employed. The two primary areas of study are DNA replication and glycosylation of lipids and proteins.

DNA Replication

The first focus of the laboratory is the mechanism and inhibition of enzymes involved in eucaryotic DNA replication. Presently, we are studying the cellular DNA polymerase alpha-primase complex, an enzyme complex required for the initiation of all new strands of DNA during S phase, andÌýHerpesÌýDNA primase, an enzyme required for initiating new strands ofÌýHerpesÌýDNA in virally infected cells. In both systems, primase synthesizes short oligoribonucleotide primers on single-stranded DNA. These primers are transferred to a DNA polymerase, either pol alpha in cells or theÌý±á±ð°ù±è±ð²õÌýDNA polymerase, and then further elongated via dNTP polymerization.

A primary interest is elucidating the detailed mechanisms of these proteins. For example, a unique feature of primase is its ability to "count" -Ìýi.e., synthesize primers of defined length regardless of template sequence. Using a combination of site-specific mutageniside, we are attempting to understand this feature. Other questions of interest include the arrangement of the primase and polymerase active sites, how the polymerase and primase communicate with and affect each other's activity, and the functional significance of the homology between the cellular primase and DNA polymerase beta.

In vivo, a large number of accessory proteins are required for DNA replication. In the pol alpha-primase system, we are extremely interested in the interactions of Replication Protein A (single-stranded DNA binding protein) with pol alpha-primase and the mechanistic consequences of these interactions. In theÌýHerpesÌýsystem, interactions between the primase, helicase and polymerase are of interest.

One of the most important aspects of enzymes biomedically is the ability to inhibit them. For example, inhibitors of enzymes involved in DNA replication are a major class of anti-cancer and anti-viral agents. Understanding how these compounds inhibit DNA replication is essential both for understanding how they are cytotoxic as well as for the design of future generations of therapeutics. Additionally, understanding how the mechanism by which various inhibitors work will provide insights into the mechanisms of the enzymes involved in DNA replication. Thus, we are both studying the mechanism by which know replication inhibitors affect these enzymes as well as synthesizing novel inhibitors. In the latter case, we hope to develop compounds that potently inhibit primase without affecting DNA polymerases.

Glycosylation

The second focus of the laboratory is lipid and protein glycosylation. We have discovered that 3'azido-3'-deoxythymidine, the primary treatment for HIV infection, is a selective and potent inhibitor of glycosylation. Inhibition is due to an AZT metabolite, AZTMP, that accumulates within cells and inhibits the import of nucleotide-sugars into the Golgi complex. Importantly, these nucleotide-sugars are the precursors for all of the glycosylation reactions that occur in the Golgi complex.

At the molecular level, we are examining the mechanism and specificity of the nucleotide-sugar translocators. Mechanistic questions include identification of the active site and understanding the basis for the enzyme acting like a "leaky antiporter". In addition to providing insights into how the translocators recognize their substrates, understanding the specificity of the nucleotide-sugar translocators is critical for developing novel inhibitors of glycosylation (see below). Questions of interest include the role of charge in substrate recognition and the portions of the nucleotide critical for binding and transport across the Golgi membrane.

The large effects of AZT on glycosylation likely cause some of the side effects associated with AZT-therapy (anemia and neutropenia). Understanding how alterations in glycosylation might elicit these effects requires knowledge of what specific glycosylation reactions are most potently affected at the cellular level. Hence, we are determining which protein and lipid glycosylation reactions are most potently inhibited by AZT.

Finally, we are designing new nucleoside-based glycosylation inhibitors that will have more powerful and specific effects on glycosylation. A number of disease states (ex. cancer), are characterized by large changes in glycosylation, and these changes in glycosylation are thought to be important for progression of these diseases. Thus, glycosylation inhibitors could provide a novel class of therapeutic agents. Classical glycosylation inhibitors, however, affect glycosylation in all cells and can result in unwanted side-effects. In contrast, it should be possible to generate nucleoside-based inhibitors that specifically affect glycosylation in only a subset of cells. This is possible because formation of the actual inhibitor inside of cells requires phosphorylation of the nucleoside by a nucleoside kinase. Importantly, these nucleoside kinases are differently regulated in different cell types.

• Steet, R. A., Melançon, P., and Kuchta, R. D. (2000) "3'-Azidothymidine Potently Inhibits the Biosynthesis of Highly BranchedÌýN-linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells"ÌýJournal of Biological Chemistry,Ìý275, 26812-26820.
• Richardson, F. C., Kuchta, R. D., Mazurkiewicz, A. & Richardson, K. A. (2000) "Polymerization of 2’-Fluoro- and 2’-O-Methyl-dNTPs by Human DNA Primase, Polymerase alpha and Polymerase gamma"ÌýBiochemical Pharmacology,Ìý59, 1045-1052.
• Steet, R., Alizebeh, M., Melançon, P., and Kuchta, R. D. (1999) "3'-Azido-3'-deoxythymidine Inhibits both the Synthesis and Shedding of Gangliosides by Melanoma Cells"ÌýGlycoconjugate Journal,Ìý16, 237-245.
• Zerbe, L.K., Goodman, M. F., Efrati, E., Kuchta, R. D. (1999) "Abasic Template Lesions Are Strong Chain Terminators for DNA Primase but not for DNA Polymerase alpha During the Synthesis of New DNA Strands"ÌýBiochemistry,Ìý38, 12908-12914.
• Arezi, B., Kirk, B. W., Copeland, W. C., Kuchta, R. D. (1999) "Interactions of DNA with Human DNA Primase Monitored with Photoactivateable Crosslinking Agents: Implications for the Role of the p58 Subunit"ÌýBiochemistry,Ìý38, 12899-12907.
• Kirk, B. W. and Kuchta, R. D. (1999) "Human DNA Primase: Anion Inhibition, Manganese Stimulation, and Their Effects onÌýin vitroÌýStart Site Selection"ÌýBiochemistry,Ìý38, 10126-10134.
• Kirk, B. W. and Kuchta, R. D. (1999) "Arg304 of Human DNA Primase Is a Key Contributor to Catalysis and NTP Binding: Primase and the Family X Polymerases Share Significant Sequence Homology"ÌýBiochemistry,Ìý38, 7727-7736.