CHARACTERIZATION OF N93S, I312T, AND A333P MISSENSE MUTATIONS IN 2 JAPANESE FAMILIES WITH MITOCHONDRIAL ACETOACETYL-COA THIOLASE DEFICIENCY

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn er ror of ketone body and isoleucine catabolisms, Japanese patients, GK01 and GK19, were found to be compound heterozygotes of 149delC and A333 P, and N93S and I312T, respectively. The latter three missense mutatio ns were individually characterized by analyses of transient expression of the cDNAs and heat stability. A333P and I312T subunits showed aber rant electrophoretic mobility on SDS PAGE. T2 protein was destabilized by A333P and existed as an insoluble form in the mitochondria. I312T mutation also destabilized T2 protein; however, some T2 protein was re tained in soluble form and reduced residual activity was apparent, N93 S mutation did not change the heat stability of T2 activity and the re duced residual activity was retained, however a considerable amount wa s observed in an insoluble form. The effects of mutations were interpr eted based on a tertiary structural model of a subunit of the human T2 , This model was constructed from the X-ray crystal structure of the h omologous peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevis iae. On the basis of this model, the positions of Ala333 and Ile312 we re far from the active site and the mutations would be expected to des tabilize the tertiary structure of T2 subunit, By contrast, Asn93 is l ocated near the active site and may function to maintain a local loop structure. The mutation of Asn93 could directly disrupt disposition of the active site. Hum Mutat 12:245-254, 1998. (C) 1998 Wiley-Liss, Inc .