An examination of the relationship between active site loop size and thermodynamic activation parameters for orotidine 5'-monophosphate decarboxylase from mesophilic and thermophilic organisms

Abstract

Closure of the active site phosphate gripper loop of orotidine 5'-monophosphate decarboxylase from Saccharomyces cerevisiae (ScOMPDC) over the bound substrate orotidine 5'-monophosphate (OMP) activates the bound substrate for decarboxylation by at least 10(4)-fold [Amyes, T. L., Richard, J. P., and Tait, J. J. (2005) J. Am. Chem. Soc. 127, 15708-15709]. The 19-residue phosphate gripper loop of the mesophilic ScOMPDC is much larger than the nine-residue loop at the ortholog from the thermophile Methanothermobacter thermautotrophicus (MtOMPDC). This difference in loop size results in a small decrease in the total intrinsic phosphate binding energy of the phosphodianion group of OMP from 11.9 to 11.6 kcal/mol, along with a modest decrease in the extent of activation by phosphite dianion of decarboxylation of the truncated substrate 1-(beta-D-erythrofuranosyl)orotic acid. The activation parameters DeltaH(double dagger) and DeltaS(double dagger) for k(cat) for decarboxylation of OMP are 3.6 kcal/mol and 10 cal K(-1) mol(-1) more positive, respectively, for MtOMPDC than for ScOMPDC. We suggest that these differences are related to the difference in the size of the active site loops at the mesophilic ScOMPDC and the thermophilic MtOMPDC. The greater enthalpic transition state stabilization available from the more extensive loop-substrate interactions for the ScOMPDC-catalyzed reaction is largely balanced by a larger entropic requirement for immobilization of the larger loop at this enzyme.

Figure 1

Comparison of the closed active site loops of the dimeric OMPDCs from S. cerevisiae (19 residues, left structure, PDB accession code 3GDL (46)) and M thermautotrophicus (9 residues, right structure, PDB accession code 3G1A (46)) liganded with 6-azauridine 5′-monophosphate. The active site loops are shown in red and the remainder of the monomer is shown in grey. The second monomer is shown in cyan (ScOMPDC) or magenta (MtOMPDC). The loop for ScOMPDC extends from Pro-202 to Val-220 and the loop for MtOMPDC extends from Pro-180 to Asp-188. The 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).

Figure 2

Dependence of (k_cat/K_m)_obsd for the decarboxylation of EO catalyzed by MtOMPDC on the concentration of added phosphite dianion at pH 7.0, 25 °C and I = 0.14 (NaCl). The closed and open symbols represent data obtained in two independent experiments that were reproducible to within 5%. The slope of the correlation gives the third-order rate constant (k_cat/K_m)/K_d = 2500 M^-1 s^-1 for the phosphite-activated reaction. The data at [HPO₃^2-] = 36 mM include error bars that indicate the estimated maximum range of experimental error (± 10%) and they were not included in the correlation (see text).

Figure 3

The temperature dependence of k_cat (s^-1) for the decarboxylation of saturating OMP catalyzed by OMPDCs from various organisms plotted according to the Eyring equation (eq 3). Key: ●, ScOMPDC; ■, EcOMPDC; ▲, MtOMPDC.

Figure 4

Correlations of ln (k_cat/K_m) with 1/T for the decarboxylation of sub-saturating OMP catalyzed by ScOMPDC (●) and MtOMPDC (■).

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