Enzymatic Mechanisms of Two Pyrimidine Decarboxylases

两种嘧啶脱羧酶的酶机制

基本信息

批准号：
6748224
负责人：
JEFFREY A SMILEY
金额：
$ 0.45万
依托单位：
YOUNGSTOWN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-01 至 2005-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Pyrimidine metabolic enzymes have long been recognized as targets in which drug intervention can be effective in slowing the growth of infectious microorganisms or rapidly proliferating cells. In two pyrimidine metabolic pathways -the de novo pathway and the thymidine salvage pathway, a rare pathway apparently present only in some fungi - the final pyrimidine base modification step to yield the uracil ring is a decarboxylation. The two decarboxylases that catalyze these reactions are OMP decarboxylase (ODCase) and iso-orotate decarboxylase (IDCase), respectively. The long-term objective of this project is to contribute to a complete elucidation of the mechanisms of these two decarboxylases, allowing a rational approach to the feasibility of these enzymes as inhibitor sites in pyrimidine metabolism. The specific aims for the study of ODCase will be to verify whether or not the active site lysine residue (Lys93 of the yeast enzyme), identified recently in crystal structures of four ODCases from different organisms, contacts O2 of the substrate, as originally postulated. This will be addressed using spectroscopic studies of ODCase in complex with various inhibitors: 1) 1H NMR spectroscopy of engineered ODCase with 15N specifically labeled at the active site amino group; 2) NMR spectroscopy of ODCase complexed with inhibitors with 15N, 13C1' and 1H1' and 3) Raman difference spectroscopy of ODCase/inhibitor complexes, to determine whether or not O2 of the pyrimidine nucleotide inhibitor is polarized by hydrogen bonding to the enzyme. The first specific aim for the study of IDCase will be to develop an effective affinity chromatography purification method based on the observed tight binding of the enzyme to 5-nitrouracil. Upon adequate enzyme purification, kinetic measurements will be determined for substrate and inhibitor binding, which may include inhibition by pharmacologically significant 5-fluorinated pyrimidines. Adequate enzyme purification will also allow examination of the mode of binding of 5-nitrouracil using NMR spectroscopy of the IDCase/[13C5] 5-nitrouracil complex. Finally, adequate protein purification will allow N-terminal amino acid sequencing, leading to the design of oligonucleotide probes that will be used in an attempt to locate the IDCase gene in a cDNA library of genes from Rhodotorula glutinis.

描述（由申请人提供）：嘧啶代谢酶长期以来被认为是药物干预可以有效的靶标减慢感染性微生物的生长或快速增殖的细胞的生长。在两种嘧啶代谢途径中 - 从头途径和胸苷打捞途径，这是一种罕见的途径，显然只存在于某种真菌中 - 最终的嘧啶基碱修饰步骤以产生尿嘧啶环为一个脱羧。催化这些反应的两个脱羧酶脱羧酶（ODCASE）和异座酸脱羧酶（IDCase）。该项目的长期目标是为完整的阐明这两个脱羧酶的机制，使得有理将这些酶作为嘧啶中的抑制剂位点的可行性方法代谢。研究ODCASE的具体目的是验证是否是否最近在来自不同生物体的四个ODCase的晶体结构，接触的O2 底物，如最初假定。这将使用光谱镜来解决与各种抑制剂复合物中的ODCase研究：1）1H NMR光谱法在活动现场氨基组中专门标记的15N的工程ODCase； 2）与抑制剂与15n，13c1'和 1H1'和3）ODCASE/抑制剂复合物的拉曼差异光谱，确定嘧啶核苷酸抑制剂的O2是否为极化通过氢键与酶结合。研究IDCase的第一个具体目的是开发有效的基于观察到的紧密结合的亲和力色谱纯化方法酶为5-硝酸的酶。有足够的酶纯化，动力学将确定针对底物和抑制剂结合的测量，这可能包括通过药理学意义的5-氟嘧啶的抑制作用。足够的酶纯化还将允许检查结合方式使用IDCASE/[13C5] 5-硝酸的NMR光谱的5-硝酸复杂的。最后，足够的蛋白质纯化将允许N末端氨基酸测序，导致设计将是寡核苷酸探针的设计用于将ID案例基因定位在来自的cDNA基因库中杜鹃谷氨酸。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: mechanistic implications.

OMP 脱羧酶反应中 O2 的显着氧同位素效应：机制意义。

DOI：
10.1039/b812979g
发表时间：
2008
期刊：
Organic & biomolecular chemistry
影响因子：
3.2
作者：
Wepukhulu,WickliffeO;Smiley,VanessaL;Vemulapalli,Bhargavi;Smiley,JeffreyA;Phillips,LindaM;Lee,JeehiunK
通讯作者：
Lee,JeehiunK

Hydrogen isotope tracing in the reaction of orotidine-5'-monophosphate decarboxylase.

乳清苷-5-单磷酸脱羧酶反应中的氢同位素示踪。