Chen S, Tomchick D R, Wolle D, Hu P, Smith J L, Switzer R L, Zalkin H
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Biochemistry. 1997 Sep 2;36(35):10718-26. doi: 10.1021/bi9711893.
De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase. An important feature of this inhibition is the fact that certain synergistic nucleotide pairs give more than additive inhibition. The physiological importance of synergism is in amplifying regulation by the adenine and guanine nucleotide end products of de novo synthesis. Using a new method to quantitate synergism, ADP plus GMP were confirmed [Meyer, E., and Switzer, R. L. (1978) J. Biol. Chem. 254, 5397-5402] to give strong synergistic inhibition of Bacillus subtilis glutamine PRPP amidotransferase. An X-ray structure of the ternary enzyme.ADP.GMP complex established that ADP binds to the allosteric A site and GMP to the catalytic C site. GMP increased the binding affinity of ADP for the A site by approximately 20-fold. Synergism results from a specific nucleotide-nucleotide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. Furthermore, synergism is enhanced by the competition between nucleotide inhibitor and PRPP substrate for the C site. Purine base specificity results from a backbone carbonyl interaction of Lys305' with the 6-NH2 group of adenine in the A site and a Ser347 Ogamma interaction with the 2-NH2 group of guanine in the C site. Steric considerations favor binding of the nucleoside diphosphate to the A site. Site-directed replacements of key residues increased the nucleotide concentrations needed for 50% inhibition and in some cases perturbed synergism. Mutations in either of the nucleotide sites perturbed function at both sites, supporting the important role of synergism.
从头嘌呤核苷酸合成至少部分受谷氨酰胺PRPP酰胺转移酶的终产物抑制调控。这种抑制的一个重要特征是某些协同核苷酸对产生的抑制作用大于相加抑制。协同作用的生理重要性在于增强从头合成的腺嘌呤和鸟嘌呤核苷酸终产物的调控作用。使用一种新的方法来定量协同作用，已证实[迈耶，E.，和斯威策，R. L.（1978年）《生物化学杂志》254，5397 - 5402]ADP加GMP对枯草芽孢杆菌谷氨酰胺PRPP酰胺转移酶有强烈的协同抑制作用。三元酶 - ADP - GMP复合物的X射线结构表明，ADP结合到变构A位点，GMP结合到催化C位点。GMP使ADP对A位点的结合亲和力增加约20倍。协同作用源于特定的核苷酸 - 核苷酸相互作用，该相互作用依赖于A位点的核苷二磷酸和C位点的核苷一磷酸。此外，核苷酸抑制剂与PRPP底物对C位点的竞争增强了协同作用。嘌呤碱基特异性源于Lys305'的主链羰基与A位点腺嘌呤的6 - NH₂基团的相互作用以及Ser347的Oγ与C位点鸟嘌呤的2 - NH₂基团的相互作用。空间因素有利于核苷二磷酸与A位点的结合。关键残基的定点置换增加了50%抑制所需的核苷酸浓度，在某些情况下扰乱了协同作用。两个核苷酸位点中的任何一个发生突变都会扰乱两个位点的功能，这支持了协同作用的重要作用。
