Investigations into the mechanism of orotidine 5'-monophosphate decarboxylase: Sources of substrate destabilization and transition state stabilization

Orotidine 5′-monophosphate decarboxylase (OMPDC) achieves a rarely paralleled rate acceleration, yet the catalytic basis prompting this enhancement have yet to be fully elucidated. To accomplish decarboxylation, OMPDC must overcome the high energy barrier due to the localized anionic charge of the intermediate. Mechanistic studies employing enzyme mutagenesis and product or intermediate analogues were used to investigate possible transition state stabilization by a carbene resonance structure. Viability of the carbene structure depends upon a key hydrogen bond between O4 of the substrate and the amide backbone of a conserved serine or threonine. Substitution of the conserved residue with Pro resulted in a kcat/KM of 1 M-1s-1; deletion of the FUMP O4 resulted in a product analogue that does not undergo H6 exchange or inhibit decarboxylation. Hence, indirect evidence reveals the O4-backbone interaction plays an important role for binding and catalysis. OMPDC likely has honed multiple mechanisms to attain its remarkable catalysis. The successful crystallizations of OMPDC a decade ago sparked hypotheses that structure and sequence conserved residues induced productive strain on the substrate-enzyme complex. Here, we demonstrate a new source of stress: a hydrophobic pocket adjacent to the OMP carboxylate that exhibits kinetic parameters characteristic of substrate destabilization. Substitution of these residues with hydrophilic side-chains, by providing hydrogen-bonding partners, decreased kcat by 10 to 10^4–fold. The same substitutions display very little change in the rate of product H6 exchange, supporting that this hydrophobic pocket affects the substrate-enzyme complex before the transition state. We also provide evidence that hydrophilic residues can insert water molecules into the pocket with detrimental effects to catalysis.

Influence of applied uniaxial stress upon the ultraviolet absorption bands in KBr and KI

The influence of uniaxial stress upon three types of imperfections occurring in the alkali halide crystal lattice has been investigated. The imperfections are the interstitial atom, the interstitial ion, and the negative ion vacancy. The interstitial atom, or H center, is a paraelastic defect which assumes a preferential crystal orientation in the field of an external mechanical stress. From the results of the reorientation kinetics - studies, it was possible to show that H centers are not stable in the KBr crystal lattice above 2SoK. At temperatures higher than 2SoK, the H centers are transformed into two new paraelastic defects, H(ii) and H(iii), possessing the same optical absorption band as the H center but differing both from the H' center and from each other in their reorientation kinetics. A study of the wavelength dependence of the H, H(ii), and VI (Na+) centers s~owed the 'existence of three similar-polarized transitions for each of these defects. One of these transitions, located at 230 run for all of the defects studied, was determined to be too high in energy to be explained by the simple X2 - level scheme. In addition, a comparison of various properties of the four defects indicates that the last three can be described as perturbed H centers. Dichroism measurements, performed as a function of temperature and wavelengt, h on the 230-nm I band in KBr, showed this band to be a composite of a band at 234 nm due to the I center and a band at 230 nm attributed to the H center. The I center dichroism was isolated and was observed under various experimental conditions. The results of these observations are consistent with a body-centered model for the I center in which the I-center absorption band is attributed to the excitation of a p-like electron on the interstitial Br- ion. Similar measurements were also perfonned on the a band in KI. The a-band dichroism measurements were found to be consistent with an electronic transition from an s-like ground state to a p-like excited state, indicating that the a center is best described as a quasi-molecule.