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.

Water quality trading: credit stacking and ancillary benefits

Ecosystems can provide many services. Wetlands, for example, can help mitigate water pollution from point sources as well as non-point sources, serve as habitat for wildlife, sequester carbon and serve as a place for recreation. Studies have found that these services can have substantial value to society. The sale of ecosystem credits has been found to be a possible way to finance construction investments in wetlands and easements to farmers to take their land out of production. At the same time, selling one ecosystem service credit may not always be enough to justify the investment. Traditionally market participants have only been allowed to sell a single credit from one piece of land, but recently there have been discussions about the possibility of selling more than one credit from a piece of land because it potentially could lead to more efficient ecosystem service provision. Selling multiple credits is sometimes referred to as credit stacking. This paper is an empirical study of the potential for credit stacking applied to the services provided by wetlands in the Upper Mississippi River Basin, specifically nitrogen, phosphorus and wildlife credits. In the setting of our study where costs are discrete rather than continuous we found that wetlands are a cost-effective way to reduce the nitrogen loads from wastewater treatment plants and that stacking nitrogen, phosphorus and wildlife credits may improve social welfare while leading to a higher level of ecosystem services. However, for credit stacking to be welfare improving we found that there needs to be a substantial demand for the credit that covers the majority of the investment in wetlands, while the credit aggregator has a choice between what ecosystem projects to undertake. If the credit that covers the majority of investment is sold first and is the sole basis of the investment decision and the objective is to improve welfare, a sequential implementation of ecosystem credits is not recommended; it would not lead to an increase in the total amount of ecosystem services provided though it would increase profit for the credit producer.