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- Zyryanov, AB, et al.
(författare)
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Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:4, s. 1065-1074
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Tidskriftsartikel (refereegranskat)abstract
- Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn2+ rather than Mg2+, the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and 11 PPases. We measured rates of PPi hydrolysis, PPi synthesis, and P-i/water oxygen exchange catalyzed by sgPPase with Mn2+, Mg2+, or Co2+ in the high-affinity metal-binding site and Mg2+ in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P-i). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PPi complex isomerization known to occur in family I PPases, (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PPi hydrolysis and product release, (c) the marked acceleration of sgPPase catalysis in the presence of Mn2+ and Co2+ results from a combined effect of these ions on bound PPi hydrolysis and P-i release, (d) sgPPase exhibits lower affinity for both PPi and P-i, and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K-m, which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PPi concentrations).
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- Edqvist, Johan, et al.
(författare)
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Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2.
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:51, s. 53544-53
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Tidskriftsartikel (refereegranskat)abstract
- This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded by SCP-X. The presented structural models of apo-AtSCP-2 and the ligand-bound conformation of AtSCP-2 reveal remarkable similarity with two of the structurally known SCP-2s, the SCP-2-like domain of human DBP and the rabbit SCP-2, correspondingly. The AtSCP-2 models in both forms have a similar hydrophobic ligand-binding tunnel, which is extremely suitable for lipid binding. AtSCP-2 showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC) from donor membranes to acceptor membranes. The transfer of BODIPY-PC was almost completely inhibited after addition of 1-palmitoyl 2-oleoyl phosphatidylcholine or ergosterol. Dimyristoyl phosphatidic acid, stigmasterol, steryl glucoside, and cholesterol showed a moderate to marginal ability to lower the BODIPY-PC transfer rate, and the single chain palmitic acid and stearoyl-coenzyme A did not affect transfer at all. Expression analysis showed that AtSCP-2 mRNA is accumulating in most plant tissues. Plasmids carrying fusion genes between green fluorescent protein and AtSCP-2 were transformed with particle bombardment to onion epidermal cells. The results from analyzing the transformants indicate that AtSCP-2 is localized to peroxisomes.
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