Catalysis by Orotidine 5鈥?Monophosphate Decarboxylase: Effect of 5-Fluoro and 4鈥?Substituents on the Decarboxylation of Two-Part Substrates

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• 作者：Bogdana Goryanova ; Krisztina Spong ; Tina L. Amyes ; John P. Richard
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：January 22, 2013
• 年：2013
• 卷：52
• 期：3
• 页码：537-546
• 全文大小：388K
• 年卷期：v.52,no.3(January 22, 2013)
• ISSN：1520-4995

文摘

The syntheses of two novel truncated analogs of the natural substrate orotidine 5鈥?monophosphate (<b>OMPb>) for orotidine 5鈥?monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(尾-d-erythrofuranosyl)-5-fluoroorotic acid (<b>FEOb>) and 5鈥?deoxy-5-fluoroorotidine (<b>5b>鈥?b>-dFOb>). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of <b>FEOb> (10 M^鈥? s^鈥?) and 1-(尾-d-erythrofuranosyl)orotic acid (<b>EOb>, 0.026 M^鈥? s^鈥?) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of <b>FEOb>. The OMPDC-catalyzed decarboxylations of <b>FEOb> and <b>EOb> are both activated by exogenous phosphite dianion (HPOb>3b>^{2鈥?/sup>), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of <b>FEOb>. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of <b>FEOb> is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in the rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of <b>EOb>, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary <b>Eb>鈥?b>FEOb>鈥POb>3b>2鈥?/sup> complex for the reaction of <b>FEOb>. The 4鈥?CHb>3b> and 4鈥?CHb>2b>OH groups of <b>5b>鈥?b>-dFOb> and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4鈥?CHb>3b> group of <b>5b>鈥?b>-dFOb> and the 4鈥?CHb>2b>OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4鈥?CHb>3b> substituent at <b>5b>鈥?b>-dFOb> is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate <b>FEOb>.}