Effects of Single Nucleotide Polymorphisms in the Human Holocarboxylase Synthetase Gene on Catalytic Activity

Authors

Shingo Esaki, University of Nebraska-LincolnFollow

Date of this Version

8-2011

Document Type

Article

Comments

A thesis submitted to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of the requirements for the Degree of Master of Science, Major: Nutrition, Under supervision of Professor Janos Zempleni. Lincoln, Nebraska: August, 2011

Copyright 2011 Shingo Esaki

Abstract

Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to carboxylases and histones in eukaryotic cells. Biotinylated carboxylases play essential roles in the metabolism of fatty acids, amino acids, and glucose; biotinylated histones play essential roles in gene regulation and genome stability. HCS null individuals are not viable whereas HCS deficiency is linked to developmental delays and phenotypes such as short life span and low stress resistance. Greater than 2,500 single nucleotide polymorphisms (SNPs) have been reported for HCS, but the biological importance of these polymorphisms is unknown. We hypothesized that some of these SNPs impair catalytic activity and that this effect can be overcome by dietary intervention with biotin. Here, we analyzed the enzyme kinetics of five recombinant HCS variants using a propionyl-CoA carboxylase surrogate (“p67”) as substrate for biotinylation. V_max of variants L216R, V96F and G510R were 6%, 78% and 73%, respectively, of the V_max in wild-type HCS. The K_m values of the variants V96F and G510R were not significantly different from wild-type HCS. The activity of L216R was too low to allow for meaningful analysis of K_m. In contrast, the affinity of variant Q699R for biotin was significantly lower than that of wild type HCS (K_m: 1.57 times that of wild type) and its V_max could be restored to that of wild-type HCS by biotin supplementation. This is the first biochemical characterization of catalytic activities of HCS variants. Also, this is the first report to show that HCS activity can be restored to normal by biotin supplementation.

Advisor: Janos Zempleni