This fellowship application is for funds to study the mechanism of two of the enzymes of the pantothenate biosynthesis, ketopantoate hydroxymethyl transferase (KPHMT) and aspartate decarboxylase (ADC). The group of Prof. Abell has cloned, overexpressed and purified both of these enzymes. The specific aims of this project are detailed in the following points: 1) Processing of the protein ADC. ADC is translated as a proenzyme which is cleaved at a Gly24-Ser 25 bond to yield an A subunit with a pyruvoyl group attached and a B subunit. The autocleavage in vitro is not chemospecific, which is unlikely to be physiologically desirable, suggesting that there are more factors involved in this processing in vivo. We plan determine to what extent the processing of the protein is related to its activity. Assaying the formation of the pyruvoyl group and enzyme activity, and correlating both of them with the amount of cleaved protein quantified by ESMS and SDS-PAGE should indicate if the activity is proportional to the processing of protein.
2) Mechanism of processing of ADC. We will attempt to ascertain this mechanism by labeling with 18 O the side chain of the serine involved in the cleavage and measuring by ESMS the distribution of 18 O between the A and B chains.
3) Mechanism of reaction of ADC. We want to study the stereochemistry of the reaction carrying it out in D 2 O and looking at the deuterium incorporation. In addition we plan to study the inhibition of this enzyme by irreversible inhibitors and its inactivation by the substrate L-aspartate.
4) Mechanistic studies on KPHMT. Little is known about the mechanism of conversion of a-ketovalerate (the substrate of KPHMT) to ketopantoate. We want to carry out a kinetic characterization of the reaction, pH-rate profile, substrate affinity, binding, etc. For this purpose we plan to use a radiochemical assay using (3-14C) a-ketovalerate. We will also study the flexibility of the enzyme with respect to the substrate to see if KPHMT could be used for the transfer of a hydroxymethyl group to different ketones.
Relevance of this proposal to Community policies.
This application is centered on the mechanistic characterization, and the utility in biotransformations of two enzymes of the pantothenate pathway. The knowledge of the structure and mechanism of these enzymes has important implications in the development of inhibitors, which could be used as herbicides or antimicrobial agents. These guidelines agree with the policy of the Community of promoting basic research, but this proposal also deals with practical applications, specially in the field of biotransformations, which is nowadays one of the priority areas of research worldwide. Furthermore this project could be also of interest to Asturias, the region in Spain where the applicant comes from, in which several groups are trying to establish research in biotransformations. In addition, this grant will help the applicant to acquire fresh knowledge in the characterization of enzymatic mechanisms by using state of the art mass spectrometry techniques, and deeper knowledge in the area of biotransformations.