Effect of naturally occurring mutations in human glycine N -methyltransferase on activity and conformation q Zigmund Luka and Conrad Wagner * Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA Received 27 October 2003 Abstract 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 hy- perbolic 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. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Glycine N -methyltransferase; Human; Mutants; Conformation; Kinetics Although first discovered in 1960 [1] and originally purified from rabbit liver in 1973 [2] most studies of glycine N -methyltransferase (GNMT) have been carried out on the enzyme from rat liver. It is believed to participate in the regulation of the ratio of S -adenosyl- methionine (AdoMet) to S -adenosylhomocysteine (AdoHcy) [2,3]. Using standard protein and molecular biology techniques it was found that GNMT is a tetra- meric protein consisting of four identical subunits of about 32.5 kDa [4,5]. Amino acid sequences of GNMT from other mammals, including human, were deter- mined by cloning and sequencing of their cDNAs [4–8]. The proteins were found to be highly similar with identity of about 90%. Each subunit of the GNMT tetramer possesses an active center where AdoMet and glycine bind [9,10]. A recombinant rat apo-protein [11,12] and a R175K mutant of GNMT in complex with AdoMet and acetate, an analog of glycine [13], were crystallized and based on the crystal structure a mech- anism of enzyme action was proposed [14]. This enzyme was re-discovered as one of the major folate-binding proteins in mammalian liver [15,16]. GNMT binds [17] and is inhibited by a form of folate, 5-methyltetrahydrofolate [16–18]. GNMT is believed to be a key component of a regulatory scheme that relates the de novo synthesis of methyl groups to the availability of pre-formed methyl groups supplied by methionine [19]. The key role of GNMT in methyl group metabolism was confirmed by the discovery of two unusual cases of per- sistent isolated hypermethioninaemia [20–22]. In both cases the levels of methionine and AdoMet in the plasma were extremely high while the levels of AdoHcy and sar- cosine were normal. It was proposed that this was a result of inactivation of GNMT [20]. By genetic and molecular biology analysis it was found that, indeed, in each case GNMT carries a missense mutation. In one case the af- fected individual is a compound heterozygote with L49P and H176N mutations [21]. In the second case the affected individual is an apparent homozygote with a N140S mutation [22]. No other mutation in the coding region was q This work was supported by Grants DK15289 and DK54859 from the US Public Health Service and from the Office of Research and Development, Medical Research Service, Department of Veteran Affairs. * Corresponding author. Fax: 1-615-343-0704. E-mail address: conrad.wagner@vanderbilt.edu (C. Wagner). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.037 Biochemical and Biophysical Research Communications 312 (2003) 1067–1072 BBRC www.elsevier.com/locate/ybbrc