Kim J H, Wolle D, Haridas K, Parry R J, Smith J L, Zalkin H
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
J Biol Chem. 1995 Jul 21;270(29):17394-9. doi: 10.1074/jbc.270.29.17394.
Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-alpha-pyrophosphoryl-2-alpha, 3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate (cPRPP). Although cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme conformation. First, cPRPP was not able to "activate" Cys1 for reaction with glutamine or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 for catalysis. Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site. However, the binding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site. Since it is expected that nucleotide inhibitors would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not activate the enzyme.
利用一种新的稳定的PRPP碳环类似物1-α-焦磷酸基-2-α,3-α-二羟基-4-β-环戊烷-甲醇-5-磷酸（cPRPP）对谷氨酰胺磷酸核糖焦磷酸（PRPP）酰胺转移酶的催化作用和调节进行了研究。尽管cPRPP与PRPP竞争结合大肠杆菌酶的催化C位点，但有两条证据表明，与PRPP不同，cPRPP不会促进酶的活性构象。首先，cPRPP无法“激活”半胱氨酸1与谷氨酰胺或谷氨酰胺亲和类似物反应。因此，PRPP的环氧化合物可能是激活半胱氨酸1进行催化的构象变化所必需的。其次，cPRPP与C位点的结合会阻止AMP和GMP（核苷酸终产物抑制剂）与该位点的结合。然而，C位点的cPRPP对变构位点上核苷酸的结合基本上没有影响。由于预计核苷酸抑制剂会以低亲和力与活性酶构象结合，因此核苷酸结合数据支持cPRPP不会激活该酶的结论。
