Anatomy of a proficient enzyme: The structure of orotidine 5′-monophosphate decarboxylase in the presence and absence of a potential transition state analog

Abstract

Orotidine 5 -phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme. The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5 -phosphate, at a resolution of 2.1 Å and 2.4 Å, respectively. Orotidine 5 -phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel. The binding of 6-hydroxyuridine 5 -phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product. In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state. Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation.O rotidine 5Ј-phosphate decarboxylase (ODCase) (EC 4.1.1.23) is responsible for de novo synthesis of uridine 5Ј-phosphate, an essential precursor of RNA and DNA. In neutral solution, orotidine 5Ј-monophosphate (OMP) undergoes spontaneous decarboxylation to uridine 5Ј-phosphate with a half-time of 78 million years (1). At the ODCase active site, the same reaction proceeds with a half-time of 18 msec (2). Comparison of k cat ͞K m with k non indicates that ODCase surpasses other enzymes in its proficiency ʈ as a catalyst, achieving a remarkable affinity for the altered substrate in the transition state (1). In addition to surmounting this formidable kinetic barrier, the ODCase reaction is of special interest in view of its lack of precedent in biological chemistry. The substrate is devoid of an effective repository for the negative charge that is generated at C-6 when CO 2 is eliminated, yet the enzyme functions without metals or other cofactors. Enzymatic decarboxylation of OMP is also remarkable in the importance (for catalysis) of a seemingly irrelevant part of the substrate. By its presence, the 5Ј-phosphoryl group contributes a factor of Ϸ10 8 -fold to k cat ͞K m , in spite of its considerable distance from the site of chemical transformation of the substrate (3). As a first step toward understanding these unusual properties, we have investigated the crystal structure of recombinant Saccharomyces cerevisiae ODCase alone and in complex with a postulated transition state analogue, 6-hydroxyuridine 5Ј-phosphate (BMP) (K i ϭ 9 ϫ 10 Ϫ12 M (4)]. Materials and MethodsRecombinant yeast ODCase was expressed in Escherichia coli SS6130 (pBGM88) and was purified as described (5). Crystals of the native enzyme were grown a...

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion

“…X-ray crystallographic analyses suggest that both TIM and OMPDC utilize the binding energy of the substrate phosphodianion group to drive the thermodynamically unfavorable closure of a mobile protein loop(s) to cover the bound phosphodianion (9)(10)(11)(12). We have proposed (8) that this loop closure sequesters the substrate from bulk solvent and results in formation of a local microenvironment in which there is optimal stabilization of transition state for the enzymatic reaction (13,14).…”

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.…”

“…16,19,30 The apparent pK a for the D71C substitution was determined to be 5.6 and that of the D76C substitution to be 6.4 (Fig. 6).…”

“…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).…”

“…17,18 The large conformation change involved moves the enzyme from an ''open'' to ''closed'' form. 17,19 It is suggested that the phosphoryl group in the substrate provides binding interactions with the loop to drive the enzyme conformational change to the closed form, which greatly stabilizes the transition state. 20,21 Thus, as has been found for other enzyme systems, relatively large conformational changes appear to be involved in the catalytic mechanism of ODCase.…”