Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding

The role of K396 in the enzymatic catalysis and the antigenicity of the 65 kDa isoform of glutamate decarboxylase (GAD65) was analyzed using the K396R GAD65 mutant, GAD65 is a major autoantigen in Type 1 diabetes and autoanti bodies directed to GAD65 are widely used markers for this disease. We found that (1) recombinant human GAD65 is fully enzymatically active; (2) the K3 96R mutation abolished GAD65 activity; and (3) the K396R mutant retained fu ll antigenicity to GAD65 autoantibodies in serum from Type 1 diabetes patie nts, but not to polyclonal antibodies raised to the catalytic domain. (C) 2 001 Federation of European Biochemical Societies. Published by Elsevier Sci ence B.V. All rights reserved.