Missense mutations in the enzyme, glycine N-methyltransferase (GNMT) have been shown to be one cause of persistent isolated hypermethioninaemia in humans. These mutations were first identified by metabolite analysis and were shown to be L49P, N140S, and H176N by gene sequencing. Here we report the kinetic and conformational characterization of the wild type and the three human mutant GNMTs expressed in Escherichia coli. Although quaternary, tertiary, and secondary structures of the mutant proteins are not changed, they are inactivated to different extents. The H176N mutation possesses 75% activity of the wild type enzyme while L49P has 10% activity and N140S has less than 0.5% activity of the wild type GNMT under the same conditions. All GNMTs display hyperbolic kinetics at neutral pH toward both substrates, S-adenosylmethionine and glycine. The turnover constants, k(cat) and Michaelis constants, K(m) for both substrates of all mutant proteins are considerably changed compared to the wild type enzyme.