Dissecting Enzyme Regulation by Multiple Allosteric Effectors: Nucleotide Regulation of Aspartate Transcarbamoylase

Abstract

The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling-high-dimensional model representation (RS-HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS-HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP × CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes.Many biochemical processes are sensitive to multiple signals, which may interact in a nonlinear manner. While such sensitivity often arises from complex reaction networks, even single enzymes can respond to multiple regulators. One such enzyme is aspartate transcarbamoylase (ATCase) (1), a complex in which activity and regulation are on distinct polypeptide chains (2). ATCase is composed of 12 polypeptides (6 c chains and 6 r chains), organized as two catalytic trimers bound to three regulatory dimers (3,4). It catalyzes the first committed step in the metabolic pathway for de novo pyrimidine biosynthesis: the condensation of aspartate with carbamoyl phosphate to yield carbamoyl aspartate. Binding of substrate to the enzyme is ordered, with carbamoyl phosphate binding before aspartate (5,6) and inducing local conformational changes leading to the formation of a viable aspartate-binding site (7). † Funding was provided by the NSF DDDAS program (CNS-0549181), the EPA STAR grant program, the DOD STTR program, the NSF CAREER award (MCB-0643859), the Beckman Foundation, the American Heart Association (0635188N), the NIH Center for Systems Biology at Princeton University (5 P50 GM071508), and NIH Grant GM26237. Both genetic and biochemical evidence support CTP, a primary end product of the pathway, being the key negative regulator of ATCase (a classical example of feedback inhibition) (8).Biochemical evidence also demonstrates that ATP substantially enhances enzyme activity (9), with this regulation serving to balance pyrimidine and purine concentrations. Crystal structures of the ATCase complex have been solved with ATP or CTP bound and confirm that ATP and CTP bind to and induce conformational changes in...

Asymmetric Allosteric Signaling in Aspartate Transcarbamoylase

“…Crystallography studies have shown that the ribose triphosphate portions of ATP and CTP bind in essentially the same part of the nucleotide-binding site. Therefore one might expect to also observe an interaction of the enzyme with other nucleotides such as UTP and GTP, although it has been reported that these nucleotides do not alter the activity of the enzyme alone or in nucleotide combinations (19). Nevertheless, UTP or GTP alone was able to drastically alter the fluorescence intensity of the HCE-Gly52 R ATCase (Figure 3, panel b ; Supplementary Table 1).…”

Vulnerability of ARID1A deficient cancer cells to pyrimidine synthesis blockade

“…ATCase, the primary regulatory enzyme of de novo pyrimidine biosynthesis, is allosterically regulated by ATP availability thereby functioning as a cellular energy sensor input to this pathway. In addition to its enzymatic activity being positively regulated by a purine (ATP), ATCase is negatively regulated by a pyrimidine (CTP), enabling its critical function of maintaining nucleotide pool balance between purines and pyrimidines 19,20 . Nucleotide pool balance is a key determinant of DNA replication fidelity.…”

Proteomic identification of responsive proteins of Vibrio parahaemolyticus under high hydrostatic pressure

“…There were also responsive proteins in the decreased manner associated with biosynthesis and cellular processes (Fig. and Table ), which included l ‐aspartate oxidase (NadB), a ubiquitous enzyme for the de novo biosynthesis of NAD + as a signalling molecule in a variety of cellular processes; Aspartate carbamoyltransferase (PyrB), a key enzyme in the first committed step of de novo pyrimidine biosynthesis that plays a critical role in cellular metabolism serving as activated precursors of RNA and DNA, CDP‐diacylglycerol phosphoglyceride for the assembly of cell membranes and UDP‐sugars for protein glycosylation and glycogen synthesis; and acetylglutamate kinase (ArgB), a member of the amino acid kinase family, which catalyses the second and frequently controlling step of arginine synthesis . Furthermore, the proteomics data revealed that the abundances of key proteins associated with transcription and translation were reduced, such as RNA polymerase sigma factor (RpoD), ribosomal proteins (RpsA and RplJ) and phenylalanyl‐tRNA synthetase (PheS) (Fig.…”