Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

Abstract

Orotidine 5 0 -monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5 0 -monophosphate to uridine 5 0 -monophosphate during pyrimidine nucleotide biosynthesis. This enzyme is one of the most proficient known, exhibiting a rate enhancement of over 17 orders of magnitude over the uncatalyzed rate. An interesting question is whether the high proficiency of ODCase is associated with a highly optimized sequence of active site residues. This question was addressed by randomizing 24 residue positions in and around the active site of the E. coli ODCase (pyrF) by site-directed mutagenesis. The libraries of mutants were selected for function from a multicopy plasmid or by single-copy replacement at the pyrF locus on the E. coli chromosome. Stringent sequence requirements for function were found for the mutants expressed from the chromosomal pyrF locus. Six positions were not tolerant of substitutions and several others accepted very limited substitutions. In contrast, all positions could be substituted to some extent when the library mutants were expressed from a multicopy plasmid. For the conserved quartet of charged residues Lys44-Asp71-Lys73-Asp76, a cysteine substitution was found to provide function at positions 71 and 76. A lower pK a for both cysteine mutants supports a mechanism whereby the thiolate group of cysteine substitutes for the negatively charged aspartate side chain. The partial function mutants such as D71C and D76C exhibit reduced catalytic efficiency relative to wild type but nevertheless provide a rate enhancement of 15 orders of magnitude over the uncatalyzed rate indicating the catalytic proficiency of the enzyme is robust and tolerant of mutation.

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

“…Orotidine 5′-monophosphate decarboxylase (OMPDC) employs no metal ions or other cofactors, but yet effects an enormous stabilization of the transition state for the chemically very difficult decarboxylation of orotidine 5′-monophosphate (OMP) to give uridine 5′-monophosphate (UMP), − by a stepwise mechanism through a UMP carbanion reaction intermediate (Scheme ). ,− OMPDC provides a large 31 kcal/mol stabilization of the transition state for the decarboxylation of OMP, and binds this transition state with a much higher affinity than substrate OMP, whose ground-state complex with OMPDC is stabilized by only 8 kcal/mol …”

Catalysis in Enzymatic Decarboxylations: Comparison of Selected Cofactor-Dependent and Cofactor-Independent Examples.

“…4) is positioned to stabilize the negative charge at C-6 and also to protonate the intermediate, which then forms the product UMP. 139 Saturation mutagenesis experiments were carried out on 24 residues of the E. coli OMPDC 140 in and around the active site. All positions could be substituted to some extent when the library mutants were expressed from a multi-copy plasmid.…”

Substrate Distortion Contributes to the Catalysis of Orotidine 5′-Monophosphate Decarboxylase

“…An experimental pK a analysis using the catalytically Journal of the American Chemical Society still active D70C and D75C mutants also supports this assumption by identifying the charge state of the cysteine side chains as thiolate ions. 13 The computational results clearly show that K42 stabilizes the ES complex more than the TS and INT structures, D70 destabilizes the ES complex in comparison to the other two states and K72 stabilizes the INT structure relative to the ES complex and TS structure. Smaller but still significant peaks are seen for several other residues but are regarded as artifacts due to the following reasons.…”

“…11,12 Random mutagenesis of E. coli chromosomal ODCase confirms that these four residues do not tolerate any substitutions. 13 In addition, quite a number of residues surrounding the substrate- binding site have also been mutated to estimate their contribution to catalysis (Figure S1), including the substrate destabilizing effect of a conserved hydrophobic patch. 14 We have extensively investigated the reaction mechanism of this enzyme by crystallographic and kinetic means.…”