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Investigation of the mechanism of quaternary structure formation of glycinamideribonucleotide transformylase

Christine Mullen

Appointment Period: 1996-1998, Grant Years: [12,13]

Christine MullenThe mechanism by which a linear sequence of amino acids folds into a single three-dimensional structure remains poorly understood. The goal of my graduate research is to further elucidate the factors responsible for the folding, stability, and activity of proteins in solution. Upon joining Dr. Patricia Jennings' lab, I chose the enzyme glycinamide ribonucleotide transformylase (GART) from the bacteria Escherichia coli as the focus of my studies. GART catalyzes the first folate-dependent step in de novo purine biosynthesis, in which a formyl group from 10-formyltetrahydrofolate is transferred to the amino group of glycinamide ribonucleotide.

Solution studies of the folate-dependent enzymes, dihydrofolate reductase and thymidylate synthase, have led to the successful design of antifolate drugs and the treatment of cancers through chemotherapy. Taken together, the fact that GART is a folate-dependent enzyme, and the fact that GART is vital to the synthesis of purine bases for DNA, make it a highly attractive candidate for the design of chemotherapeutic drugs.

In preparation for folding studies, I first probed the enzyme structure spectroscopically as a function of the solution pH and ionic strength. In the pH range 7.5 to 6.8, monomeric GART reversibly associates into a dimeric form. Furthermore, as GART dimerizes, the monomer undergoes several structural changes. Presently, there are several published structures of the enzyme, all dimeric, which are being used as models for drug design. The fact that the enzyme activity is maximal under conditions where GART is both fully monomeric and structurally different from its dimeric form makes it imperative that drugs be designed against the monomer. An additional consequence of my studies is the distinct possibility that GART may be regulated via subtle cellular pH changes.

PUBLICATIONS (resulting from this training)

Mullen CA, Jennings PA. (1996) Glycinamide ribonucleotide transformylase undergoes pH-dependent dimerization. J Mol Biol. 262:746-55.

Mullen CA, Jennings PA. (1998) A single mutation disrupts the pH-dependent dimerization of glycinamide ribonucleotide transformylase. J Mol Biol. 276:819-27.

Su Y, Yamashita MM, Greasley SE, Mullen CA, Shim JH, Jennings PA, Benkovic SJ, Wilson IA. (1998) A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotidetransformylase at 1.8 to 1.9 A. J Mol Biol. 281:485-99.