| 1. |
- Phillips, R S, et al.
(författare)
-
Role of Lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation
- 2004
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:45, s. 14412-14419
-
Tidskriftsartikel (refereegranskat)abstract
- Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K+ or NH4+, for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K-m values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K-m about 10% of wild-type TPL), is activated by K+, and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-CYS or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain I mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.
|
|
| 2. |
- Vernant, P, et al.
(författare)
-
Deciphering oblique shortening of central Alborz in Iran using geodetic data
- 2004
-
Ingår i: Earth and Planetary Science Letters. - : Elsevier. - 0012-821X. ; 223:1-2, s. 177-185
-
Tidskriftsartikel (refereegranskat)abstract
- The Alborz is a narrow (100 km) and elevated (3000 m) mountain belt which accommodates the differential motion between the Sanandaj–Sirjan zone in central Iran and the South Caspian basin. GPS measurements of 12 geodetic sites in Central Alborz between 2000 and 2002 allow to constrain the motion of the belt with respect to western Eurasia. One site velocity on the Caspian shoreline suggests that the South Caspian basin moves northwest at a rate of 6±2 mm/year with respect to western Eurasia. North–South shortening across the Alborz occurs at 5±2 mm/year. To the South, deformation seems to extend beyond the piedmont area, probably due to active thrusting on the Pishva fault. We also observe a left-lateral shear of the overall belt at a rate of 4±2 mm/year, consistent with the geological motion observed along E–W active strike-slip faults inside the belt (e.g., the Mosha fault).
|
|
