Redesign of Alpha Class Glutathione Transferases to Study Their Catalytic Properties

Nilsson, L. O. 2001. Redesign of Alpha Class Glutathione Transferases to Study Their Catalytic Properties. Acta Univ. Ups. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 672. 30 pp. Uppsala. ISBN 91-554-5171-3. A number of active site mutants of human Alpha class glutathione transferase A1-1 (hGST A1-1) were made and characterized to determine the structural determinants for alkenal activity. The choice of mutations was based on primary structure alignments of hGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, from three different species and crystal structure comparisons between the human enzymes. The result was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1chloro-2,4-dinitrobenzene (CDNB). The C-terminus of the Alpha class enzymes is an -helix that folds over the active site upon substrate binding. The rate-determining step is product release, which is influenced by the movements of the C-terminus, thereby opening the active site. Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine 9 and phenylalanine 10, positioning the -carbon of the cysteinyl moiety of glutathione. The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studied by the viscosity dependence of kcat and kcat/Km with glutathione and CDNB as the varied substrates. The compatibility of slightly different subunit interfaces within the Alpha class has been studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4. The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 C. The heterodimers did not show strictly additive activities with alkenals and CDNB. This result combined with further studies indicates that there are factors at the subunit interface influencing the catalytic properties of hGST A1-1. Lisa O. Nilsson, Department of Biochemistry, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden Lisa O. Nilsson 2001-10-19 ISSN 1104-232X ISBN 91-554-5171-3 Printed in Sweden by Eklundshofs grafiska, Uppsala 2001