Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization^,

Abstract

The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k cat /k non , 7.1 × 10 16 ) and proficiency [(k cat /K M )/k non , 4.8 × 10 22 M −1 ] is uncertain. That the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and . We investigated that hypothesis by structural and functional characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the value of k cat for decarboxylation of FOMP (a more reactive substrate analog) but have little effect on the value of k ex for exchange of H6 of FUMP with solvent deuterium; the structures of wild type MtOMPDC and its mutants are superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild type ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP, with differences in both the conformation of the active site loop and the orientation of the ligand vis á vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization. † This research was supported by NIH GM039754 (to J.P.R.) and GM065155 (to J.A.G.). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). The X-ray coordinates and structure factors for the following structures have been deposited in the Protein Data Bank with the indicated accession codes: wild type MtOMPDC in the absence of ligands (PDB accession code 3G18), wild type MtOMPDC complexed with 6-azaUMP (3G1A), wild type MtOMPDC complexed with UMP (3G1D), wild type MtOMPDC complexed with H 2 OMP (3D1F), wild type MtOMPDC complexed with H 2 UMP (3D1H), the D70N mutant of MtOMPDC in the absence of ligands (3G1Y), the D70N mutant of MtOMPDC complexed with 6-azaUMP (3G24), the D70N mutant of MtOMPDC complexed with UMP (3G22), the D70G mutant of MtOMPDC in the absence of ligands (3G1S), the D70G mutant of MtOMPDC complexed with UMP (3G1X), the D70G mutant of MtOMPDC complexed with FOMP (3G1V), wild type ScOMPDC in the absence of ligands (3GDK), wild type ScOMPDC complexed with 6-azaUMP (3GDL), the D91N mutant of ScOMPDC in the absence of ligands (3GDR), and the D91N mutant of ScOMPDC complexed with 6-azaUMP (3GDT).

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

“…The group of charged residues including Lys44-Asp71-Lys73-Asp76 is of particular interest, because they are thought to play a key role in catalysis. 16,19,29,30 It has been shown with the yeast ODCase that alanine substitutions at positions equivalent to Lys73, Asp71, and Asp76 result in at least a 10 5 fold loss in enzyme specific activity in each case, which is consistent with a critical role of these residues in catalysis. 29 In addition, substitution of the Lys44 equivalent residue with alanine resulted in an $ 10 3 -fold loss in specific activity.…”

“…Others have previously performed site-directed mutagenesis studies of active site residues, in particular the group of charged residues including Lys44-Asp71-Lys73-Asp76. 16,19,29,30 However, there has been no previous site-saturation random mutagenesis performed and also no attempts to systematically mutagenize all residues in and proximal to the active site. The 24 residues chosen for randomization were picked based on their location relative to the barbituric acid monosphosphate (BMP) inhibitor bound in the E. coli ODCase active site crystal structure (PDB code: 1EIX).…”

“…5). 11,16 Because of the importance of these positions in the catalytic mechanism, it was expected that only aspartate would be selected for function among random mutants at positions 71 and 76. Consistent with expectations, aspartate was found most often among functional mutants from the 71 and 76 libraries (Fig.…”

“…A D70N (D71N E. coli) substitution in the Mt ODCase results in an 840-fold decrease in k cat /K m and a D96A (D76A E. coli) substitution in yeast ODCase results in a 10 5 -fold loss in enzyme specific activity measured in vitro. 16,29 It is possible that the in vivo selection requires only low levels of ODCase activity for growth of E. coli on M9 minimal medium or, alternatively, the cysteine substitutions may provide high level activity for the E. coli enzyme.…”

“…[13][14][15] Recent results indicate that Asp70 in the Methanothermobacter thermautotrophicus (Mt) and the analogous Asp91 in the yeast enzyme (E. coli Asp71) provide electronic repulsion and steric hindrance with the carboxylate group to destabilize substrate. 16 This positioning then enables Lys 72 (Mt) and Lys93 (yeast) (E. coli Lys73) to interact preferentially with the negative charge forming at C6 in the transition state. The anionic transition state is proposed to be stabilized by the positive charge and then protonated by this active site lysine.…”