DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project involves the study of a critical enzyme in metabolism, aspartate transcarbamoylase (ATCase). ATCase catalyzes the first step in pyrimidine nucleotide biosynthesis. The product of the reaction, carbamoyl aspartate is then converted into the pyrimidine nucleotides necessary for nucleic acid biosynthesis. ATCase has been identified as a target for anti-proliferation and anti-malarial drugs. Particularly important is that ATCase not only catalyzes the above reaction, but also controls the rate of pyrimidine biosynthesis. Regulation is achieved by a conformational switch from a low-activity T-state to a high-activity R-state. These two states have different quaternary conformations that can be easily distinguished by SAXS. By using a stopped flow mixer attached to the SAXS apparatus at SSRL we are ability to monitor the actual transition of the enzyme from the T to the R, and from the R to T, states induced by the natural substrates as well as potential drug candidates. For this project period we have two specific aims: (i) investigate the heterotropic interactions and homotropic cooperativity of ATCase using time-resolved SAXS, and (ii) monitor the cooperativity transition of ATCase from the T to the R state by time-resolved crystallography. The first specific aim is directed at determining the molecular level details of how ATCase is able to regulate pyrimidine nucleotide biosynthesis. The second specific aim will utilize the new capabilities of beamline 4-2 to obtain a time-lapsed record of the conformational changes that are required to convert the enzyme from the T to the R state by x-ray crystallography. This experiment will then be combined with other ongoing studies to determine by crystallography each of the steps in the catalytic and regulatory mechanisms of this important metabolic enzyme.