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Human liver glycolate oxidase: gene identification and protein studies

Williams, EL; (2003) Human liver glycolate oxidase: gene identification and protein studies. Doctoral thesis (PhD), UCL (University College London). Green open access

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Glycolate oxidase (GO) is a peroxisomal flavoenzyme which catalyses the oxidation of short chain a-hydroxy acids, notably glycolate. The reaction product, glyoxylate, is an oxalate precursor and GO is thus of potential interest for its role in the pathogenesis of the primary hyperoxalurias. The project aims were to identify human GO, characterise the kinetics and substrate specificity of the enzyme and establish methods for the analysis of relevant metabolic pathways in vitro. The gene for human GO was cloned from liver and expressed in bacterial cells. The cDNA is 1128 bp in length and has a 1113 bp open reading frame encoding a 372 amino acid protein. The genomic sequence comprises eight exons and spans —57 kb of chromosome 20p12. Recombinant human GO protein shares 53% and 89% sequence similarity to GO from spinach and rat respectively, shows a-hydroxy acid oxidase activity in vitro and has been purified to homogeneity. Polyclonal anti-GO antibody detects a band of 43 kDa in human liver and, consistent with northern blot analysis, expression is not detected in other tissues including kidney and leucocytes. Kinetic analysis with a range of a-hydroxy acids indicates GO has highest affinity for glycolate as substrate (Km = 0.54 mM) and 10 fold less affinity for glyoxylate (Km = 5.1 mM). Site directed mutagenesis of active site residues demonstrates the importance of chain length for substrate affinity. Thus mutation of a Trp residue, conserved between spinach and human GO to a less bulky amino acid, permits the catalysis of longer chain length a-hydroxy acids. HPLC methods were developed for the separation and quantitation of glyoxylate, hydroxypyruvate and pyruvate, enabling analysis of metabolites produced by GO and neighboring enzymes in the metabolic pathway. These assays will be invaluable for future studies in which the pathways of glyoxylate metabolism are constructed in vitro.

Type: Thesis (Doctoral)
Qualification: PhD
Title: Human liver glycolate oxidase: gene identification and protein studies
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Third party copyright material has been removed from ethesis.
URI: https://discovery.ucl.ac.uk/id/eprint/10057809
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