Mechanism of decarboxylation of 1,3-dimethylorotic acid. A model for orotidine 5'-phosphate decarboxylase

Abstract

with stirring, under nitrogen at 5 OC, 0.25 ml of methyl fluorosulfonate (3.1 mmol) over a period of 15 min. The oily precipitate was stirred at 4 OC overnight to give a white solid, which was washed repeatedly with dry ether and dried under vacuum over P205. The slightly hydroscopic material decomposed in the range 60-80 OC. The NMR spectrum in CD3CN gave the following peaks relative to Me&: 6 9.1 ( 1 H, s), 7.9-7.5 ( 7 H, m), and 4.0 (3 H, s). This material was used in the kinetic studies without further purification.Kinetic Measurements, Pseudo-first-order rate constants were determined either spectrophotometrically at 260 nm using a Cary 14 spectrophotometer or by a pH stat method using a Radiometer apparatus which included a TTT60 titrator, PHM62 pH meter, ABU 12T buret, and a REC61 servograph. Stock solutions of 3 were prepared in acetonitrile and were stable for weeks when stored at -15 "C. Stock solutions of 4 were prepared in acetonitrile and used the same day. Stock solutions of 2 were prepared in MezSO and were used within 2 h. No solvent was found for 2 which afforded solutions stable for more than 2 h. Typically, 10-50 p1 of stock solution was used to initiate the reaction. In many pH-stat runs, 2 was added as a solid directly to the reaction vessel. Results using this technique compared well with results using fresh stock Me2SO solutions. The concentration of substrates was approximately 3 X M for the pH-stat runs and about 2.5 X M for the spectrophotometric runs. The pseudofirst-order rate constants were fit to the appropriate rate expression using a nonlinear least-squares computer program.Abstract: The decarboxylation of 1,3-dimethylorotic acid (1) is shown to proceed by separate pH-determined pathways in sulfolane at 180-220 OC. Although a process involving ionization of 1 is the major pathway in the presence of excess base, decarboxylation is initiated by zwitterion formation in the neutral solvent. Measurements of the rate of loss of carbon dioxide from 6-carboxy-2,4-dimethoxypyrimidine ( 5 ) and 1 -methyl-2,4-dimethoxypyrimidinium-6-carboxylate betaine (7) are used to estimate the equilibrium and rate constants for the zwitterionic pathway. Comparison of the rate constant for decarboxylation of 7 with k,,, for orotidine 5'-phosphate decarboxylase shows that the biological catalysis can be satisfactorily accounted for if the enzyme provides a site which displaces the equilibrium in favor of the zwitterionic form of orotidylic acid. It is also noted that the inhibitor 6-azauridine monophosphate, which has a greater affinity for the enzyme than does the substrate, provides a partial model for the intermediate formed on loss of carbon dioxide from the zwitterion.

Stability of the 6-Carbanion of Uracil Analogues: Mechanistic Implications for Model Reactions of Orotidine-5‘-monophosphate Decarboxylase

“…6,7 As a result, a two-step mechanism has been proposed to account for the large difference in rate constants measured for acids 1, 4, and 5 (Scheme 2). 1,7 In this mechanism, the large differences in the equilibrium constants explain the differences in rate constants.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12] Most of the studies involve the investigation of the nature and stability of the intermediate. As shown in Scheme 1, acid 1 decarboxylates at elevated temperatures to give 1,3-dimethyluracil (2) as the sole product.…”

“…6,7 As a result, a two-step mechanism has been proposed to account for the large difference in rate constants measured for acids 1, 4, and 5 (Scheme 2). 1,7 In this mechanism, the large differences in the equilibrium constants explain the differences in rate constants.The pK a of uracil 2 in water has been determined to be 34 ± 2, which is suggested to be the evidence for the high reaction barrier for catalysis by ODCase. 8 However, our gas-phase study has demonstrated that the stability of the carbanionic intermediates does not correlate with the rate of decarboxylation.…”

Electrostatic stress in catalysis: Structure and mechanism of the enzyme orotidine monophosphate decarboxylase

“…The ODCase reaction is unique in biological decarboxylation reactions in that the carbanion intermediate is not stabilized by conjugation interactions and, thus, the reaction rate is exceptionally slow in aqueous solution (2). The remarkable catalytic power of ODCase, which accelerates the reaction by 17 orders of magnitude over the aqueous process, has fascinated chemists and biochemists alike, leading to a number of proposals of mechanisms with novel features (3)(4)(5)(6)(7). However, as more results accumulated for this class of enzymes, possibilities for the mechanism became increasingly limited as cofactors and catalytic groups continued to be excluded from consideration (8)(9)(10).…”

Crystallization of native and selenomethionyl yeast orotidine 5′-phosphate decarboxylase

“…The spontaneous loss of CO 2 from OMP is an extremely slow reaction that proceeds with a half-time of nearly 7.8 Â 10 7 y in aqueous solutions at 298 K (Radzicka & Wolfenden, 1995). Despite numerous studies involving model decarboxylation reactions (Beak & Siegel, 1976;Silverman & Groziak, 1982;Lee & Houk, 1997), the mechanism employed by ODCase remains a mystery. In contrast to other enzymatic decarboxylations, ODCase is unique in that it appears to function independently of known cofactors and metals (Shostak & Jones, 1992;Miller et al, 1999) while enhancing the spontaneous rate of OMP decarboxylation by nearly 17 orders of magnitude.…”