Thesis (Ph. D.)--Virginia Polytechnic Institute, 1964. / Vita. Abstract. Includes bibliographical references (leaves 29-30). Also available via the Internet.

Thesis advisor: Evan R. Kantrowitz / For nearly 60 years the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and 5 X-ray structures determined in the absence and presence of a Mg2+ concentration within the physiological range. In the presence of 2 mM divalent cations (Mg2+, Ca2+, Zn2+) CTP does not significantly inhibit the enzyme while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor in vivo of ATCase is the combination of CTP, UTP and a M2+ cation and the actual allosteric activator is ATP and M2+ or ATP, GTP and M2+. The structural data reveals that two NTPs can bind to each allosteric site with a Mg2+ ion acting as a bridge between the triphosphates. Thus the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal the catalytic chains undergo essentially no alternations, however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal regions of the regulatory chains exist in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed. / Thesis (MS) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The factors which regulate enzyme activity in the growing cell have been the subject of active research during the past decade and are generally considered under the heading of "metabolic control". At least three control parameters must be considered as regulating the active level of a given enzyme: control of enzyme synthesis at the gene level, control of enzyme activity by small molecule metabolites once the enzyme has been synthesized, and control of enzyme activity imposed by its structural orientation within the cell and/or its organelles (which would affect permeability of substrates, cofactors, etc.). It can be hypothesized that periodism in the intracellular level of a given metabolite must be accompanied by periodism in the active level of one or more of the enzymes responsible for the synthesis or breakdown of this metabolite. Synchronized cultures of microorganisms afford a unique tool for studying periodic changes in the intracellular levels of metabolites during cell growth. The studies presented in this thesis were designed to elucidate the relationships between the activity of Aspartate Transcarbamylase and factors which affect and are affected by the activity of this enzyme in synchronized cultures of Chlorella pyrenoidosa. Aspartate Transcarbamylase, the first enzyme in pyrimidine biosynthesis, has been reported to be controlled by a phenomenon known as " product-inhibition". This enzyme was located in the soluble supernatant of a 100,000 x g preparation of sonicated C. pyrenoidosa ceils. The pH optimum (9.2), temperature optimum (approx. 37°), and stability characteristics of this enzyme from this organism are reported. Aspartate Transcarbamylase when measured during two consecutive synchronous growth cycles in C. pyrenoidosa under continuous illumination was found to increase at alternating exponential rates in each growth cycle. When the increase in Aspartate Transcarbamylase was expressed in its logarithmic form, a plot with linear segments was obtained, each segment having a different slope. The rate of increase of Aspartate Transcarbamylase activity during early daughter cell development changes (increases) at the 5th h. The 5th through 9th h of cell growth, where Aspartate Transcarbamylase activity is increasing at an accelerated rate, corresponds to the premitotic and nuclear division stages in the cell. Correlated with this period of increased Aspartate Transcarbamylase activity (5th through 9th h) there is an increase in the rate of accumulation of RNA and DNA concurrent with increase in the rate of accumulation of intermediates in the acid-soluble, Norit-A adsorbable-P pool (which contains the nucleotide-P fraction). At. approximately the 9th h of cell growth there is a decrease in the rate of increase of Aspartate Transcarbamylase activity. Evidence is presented indicating that the depression of activity during this period is at least partly due to the presence of Norit-A adsorbable compound(s) present in the cell (pyrimidine nucleotides, the principal inhibitors of this enzyme, would be Norit-A adsorbable). At the 8th h of cell growth ( 1h prior to the period of depressed Aspartate Transcarbamylase activity) the acid-soluble, Norit-A adsorbable-P pool reaches a maximum value (as % of total cellular-P). These data together with inhibition studies with a variety of nucleoside mono-, di-, and tri-phosphates suggest that pyrimidine nucleotides may be factors regulating Aspartate Transcarbamylase activity during cellular development. / Ph. D.