Regulation and catalysis of glutamine phosphoribosylpyrophosphate amidotransferase

Sihong Chen, Purdue University

Abstract

Glutamine PRPP amidotransferase (GPATase) catalyzes the first committed reaction of de novo purine pathway and is the key regulatory enzyme in the pathway. Four projects were carried out to study the regulation and reaction mechanism of GPATase. In the first project, the regulation of GPAT gene expression was studied in human cells. A bi-directional promoter that promotes both GPAT and AIRC gene expression was analysed in vivo and in vitro . A transcription factor that plays role in both gene expressions was purified and identified as nuclear respiratory factor 1 (NRF-1), suggesting that human de novo purine pathway may be co-regulated with mitochondria respiratory machinery that provides energy for synthesis of purine nucleotides. In the second project, the regulation of GPATase by end product inhibition was studied. The mechanism of nucleotide synergistic inhibition of B. subtilis GPATase was defined. ADP and GMP are the strongest synergistic inhibition pair that specifically binds to the allosteric and PRPP active sites of the enzyme respectively. The nucleotide synergistic inhibition was due to synergistic binding, resulting from direct interaction between two nucleotides at each site. This inhibition accounts for the physiological control of adenine and guanine nucleotide production. In the third project, GPATases having FeS cluster are also regulated by O2 dependent inactivation as a result of destruction of FeS cluster in GPATase. The incorporation of an FeS cluster into B. subtilis GPATase was studied. NifS plays a role in FeS assembly. Co-expression of NifS with B. subtilis GPATase containing an FeS cluster in an E. coli strain increased the amount of active enzyme expression. In the fourth project, the reaction mechanism of E. coli GPATase was studied. To investigate enzyme conformational changes required for catalysis in solution, a single tryptophan reporter system is established. Trp fluorescence not only reported the sequential conformational transition correlated with enzyme catalysis but also detected an intermediate that is required for enzyme reaction using external NH3 as nitrogen source (NH3-PRA reaction). Pre-steady state kinetics study further defined the pattern and rate of conformational change. The rate of conformational change induced by PRPP is the rate-limiting step for tile NH3-PRA reaction.

Degree

Ph.D.

Advisors

Zalkin, Purdue University.

Subject Area

Molecular biology|Biochemistry

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