Small GTPases (original) (raw)
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Biochemical Journal, 1996
We have examined the functional importance of the cysteine residues of rat liver S-adenosylmethionine synthetase. For this purpose the ten cysteine residues of the molecule were changed to serines by site-directed mutagenesis. Ten recombinant enzyme mutants were obtained by using a bacterial expression system. The same level of expression was obtained for the wild type and mutants, but the ratio of S-adenosylmethionine synthetase between soluble and insoluble fractions differed for some of the mutant forms. The immunoreactivity against an anti-(rat liver S-adenosylmethionine synthetase) antibody was equivalent in all the cases. Effects on S-adenosylmethionine synthetase activities were also measured. Mutants C57S, C69S, C105S and C121S showed decreased relative specific activity of 68, 85, 63 and 29%, respectively, compared with wild-type, whereas C312S resulted in an increase of 1.6-fold. Separation of tetramer and dimer forms for wild type and mutants was carried out by using phen...
Structure and function study of the complex that synthesizes S -adenosylmethionine
IUCrJ, 2014
S-Adenosylmethionine (SAMe) is the principal methyl donor of the cell and is synthesizedviaan ATP-driven process by methionine adenosyltransferase (MAT) enzymes. It is tightly linked with cell proliferation in liver and colon cancer. In humans, there are three genes,mat1A, mat2Aandmat2B, which encode MAT enzymes.mat2Aandmat2Btranscribe MATα2 and MATβ enzyme subunits, respectively, with catalytic and regulatory roles. The MATα2β complex is expressed in nearly all tissues and is thought to be essential in providing the necessary SAMe flux for methylation of DNA and various proteins including histones. In human hepatocellular carcinomamat2Aandmat2Bgenes are upregulated, highlighting the importance of the MATα2β complex in liver disease. The individual subunits have been structurally characterized but the nature of the complex has remained elusive despite its existence having been postulated for more than 20 years and the observation that MATβ is often co-localized with MATα2. Though SA...
A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl J-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 300% of the total bacterial protein.
How is rat liver S-adenosylmethionine synthetase regulated?
FEBS Letters, 1992
The in vivo regulation of S-adenosylmethionine synthetase, a key enzyme in methionine metabolism, is so far unknown. The enzyme activity has been shown to be modulated by glutathione and the oxidation state of its sulfhydryl groups. Analysis of the protein sequence has revealed the presence of putative phosphorylation sites. A mixed regulatory mechanism combining phosphorylation and the oxido/reduction of sulfhydryl groups is proposed. The role of glutathione in this mechanism is also discussed.
Biochemistry, 1990
The entire family of A T P analogues that are either mono-or disubstituted with imido and methylene bridges in the polyphosphate chain of ATP have been investigated as substrates and inhibitors of S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase). The disubstituted analogues adenosine S-(a,P:P,y-diimidotriphosphate) (AMPNPNP) and adenosine S-(a,P:a,/3'-diimidotriphosphate) [AMP(NP),] have been synthesized for the first time, and a new route to adenosine 5'-(a,/3$,y-dimethylenetriphosphate) (AMPCPCP) has been developed. S-Adenosylmethionine synthetase catalyzes a two-step reaction: the intact polyphosphate chain is displaced from ATP, yielding AdoMet and tripolyphosphate, followed normally, but not obligatorily, by the hydrolysis of the tripolyphosphate to pyrophosphate and orthophosphate. Uniformly, the imido mono-or disubstituted derivatives are both better substrates and better inhibitors than their methylene counterparts. AMPNPNP reacts rapidly to give a single equivalent 'This work was supported by U S . Public Health Service Grants GM-39552 and AR-17323 (to G.L.K.), GM-31186 (to G.D.M.), and CA-06927 and RR-05539 (to the Fox Chase Cancer Center) and also an appropriation from the Commonwealth of Pennsylvania.
S-adenosylmethionine and proliferation: new pathways, new targets
Biochemical Society Transactions, 2008
SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.
Structure-function relationships in methionine adenosyltransferases
Abstract. Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified.
Structural aspects on rat liver S-adenosylmethionine synthesis
2003
Methionine adenosyltransferase is the enzyme that catalyses the synthesis of S-adenosylmethionine. In mammals three isoenzymes have been purified, two of which, MAT III and I, are products of the same gene MAT1A, but differ in their kinetic constants and oligomerization state. Changes in activity and MAT I/III ratio have been detected in animal models and in human alcohol liver cirrhosis, leading to an increasing interest in the mechanisms that control the protein association state. In this line, data demonstrating the importance of sulfhydryl groups on the activity and oligomerization of the protein have been obtained. These results along with knowledge of the crystal structure of MAT I and the establishment of an overall-folding pathway allow an integrated view for MAT behaviour.