Tyr71 is an invariant residue in all known sequences of tyrosine phenol-lyase (TPL). The substitution of Tyr71 in TPL by phenylalanine results in a mutant Y71F TPL with no detectable activity (greater than 3 x 10(5)-fold reduction) for beta-elimination of L-tyrosine. Y71F TPL can react with S-alkylcysteines, but these substrates exhibit kcat values reduced by 10(3)-10(4)-fold, while the kcat/Km values are reduced by 10(2)-10(3)-fold, compared to wild-type TPL. However, for substrates with good leaving groups (S-(o-nitrophenyl)-L-cysteine,beta-chloro-L-alanine, and O-benzoyl-L-serine), Y71F TPL exhibits kcat values 1.85-7% those of wild-type TPL. Y71F TPL forms very stable quinonoid complexes with strong absorbance at 502 nm from L-phenylalanine, tyrosines (L-tyrosine, 3-fluoro-L-tyrosine, and [alpha-2H]-3-fluoro-L-tyrosine), and S-alkylcysteines (S-methyl-L-cysteine, S-ethyl-L-cysteine, and S-benzyl-L-cysteine). The time courses of the formation of quinonoid intermediates in these reactions are biphasic. The slow phase shows a dependence on concentration of PLP and is due to the cofactor binding steps, while the fast phase is due to the amino acid alpha-deprotonation and reprotonation steps. The rate constants for the fast phase of the reactions of Y71F TPL with L-phenylalanine and S-methylcysteine are similar to those for alpha-deprotonation or reprotonation steps in the reactions of wild-type TPL. The PLP binding constant of Y71F TPL is estimated to be 1 mM by spectrophotometric titration, compared to 0.6 microM for wild-type TPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is a pyridoxal 5′-phosphate (PLP)dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Tyr to give phenol and ammonium pyruvate. The proposed reaction mechanism for TPL involves formation of an external aldimine of the substrate, followed by deprotonation of the R-carbon to give a quinonoid intermediate. Elimination of phenol then has been proposed to give an R-aminoacrylate Schiff base, which releases iminopyruvate that ultimately undergoes hydrolysis to yield ammonium pyruvate. Previous stopped-flow kinetic experiments have provided direct spectroscopic evidence for the formation of the external aldimine and quinonoid intermediates in the reactions of substrates and inhibitors; however, the predicted R-aminoacrylate intermediate has not been previously observed. We have found that 4-hydroxypyridine, a non-nucleophilic analogue of phenol, selectively binds and stabilizes aminoacrylate intermediates in reactions of TPL with S-alkyl-L-cysteines, L-tyrosine, and 3-fluoro-L-tyrosine. In the presence of 4-hydroxypyridine, a new absorption band at 338 nm, assigned to the R-aminoacrylate, is observed with these substrates. Formation of the 338 nm peaks is concomitant with the decay of the quinonoid intermediates, with good isosbestic points at ∼365 nm. The value of the rate constant for aminoacrylate formation is similar to k cat , suggesting that leaving group elimination is at least partially rate limiting in TPL reactions. In the reaction of S-ethyl-L-cysteine in the presence of 4-hydroxypyridine, a subsequent slow reaction of the R-aminoacrylate is observed, which may be due to iminopyruvate formation. Both L-tyrosine and 3-fluoro-L-tyrosine exhibit kinetic isotope effects of ∼2-3 on R-aminoacrylate formation when the R-2 H-labeled substrates are used, consistent with the previously reported internal return of the R-proton to the phenol product. These results are the first direct spectroscopic observation of R-aminoacrylate intermediates in the reactions of TPL.

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), 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 1 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.

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Background Percutaneous nephrolithotomy (PCNL) is a safe and efficient treatment for intro-renal diseases, most of which are calculus disease. In this study, we carried out percutaneous endoscopic nephron-sparing ablation for renal carcinoma in carefully selected patients. Our aim was to evaluate whether percutaneous endoscopic nephron-sparing operation was feasible for patients with renal cell carcinoma.Methods A total of 15 patients with renal pelvis carcinoma were treated with laser evaporation under percutaneous endoscopy between January 2015 and September 2019 ( group I ). Another 13 patients who received standard radical nephroureterectomy were recruited as the control group (group II). We recorded demographic data of the patients, the indication for surgery, tumor pathological grade, size and side of tumor, and the intraoperative and postoperative outcome, including the duration of surgery, length of hospital stay, and complication rate, as well as progression-free survival (PFS).Results In both groups, all of the patients received flexible ureteroscopy. In group I, 14 patients had transitional renal cell carcinoma (UCC), four had pathological grade I, nine had grade II, and one had hemangiopericytoma. In group II, all patients had UCC, five had pathological grade I, and eight had grade II. In group I, the mean operation time was 118 min (65–236 min), the mean blood loss was 110 ml (55–220 ml), and the mean hospital stay was 9 days (7–12 days). During follow-up, two patients died and two had recurrence; among them, one had systematic bone metastasis. The PFS rate was 66.7% (10/15). In group II, the mean operation time was 265 min (185–436 min), the mean blood loss was 133 ml (85–240 ml), and the mean hospital stay was 13 days (9–16 days). During the follow-up, two patients died and there was no local or systematic metastasis. The PFS rate was 84.6% (11/13). There was no significant difference in blood loss between the groups. However, the operation time and mean hospital stay were significantly shorter in group I compared with that in group II ( P < 0.05). But the PFS rate was significantly higher in group II than in group I (P < 0.05).Conclusions This study shows that laser evaporation under percutaneous endoscopy for renal pelvis carcinoma is a safe, effective, and technically feasible procedure for treating benign and malignant renal pelvis carcinoma.

Spherical cap harmonic (SCH) theory has been widely used to format regional model of fields that can be expressed as the gradient of a scalar potential. The functions of this method consist of trigonometric functions and associated Legendre functions with integral-order but non-integral degree. Evidently, the constructing and computing of Legendre functions are the core content of the spherical cap functions. In this paper,the approximated calculation method of the normalized association Legendre functions with non-integral degree is introduced and an analysis of the entire order of associated non-Legendre function calculation is presented. Besides, we use the Muller method to search out for all intrinsic values. The results showed that the highest order of spherical harmonic function for constructing regional model of fields is limited, thus high-resolution spherical harmonic structure of local gravity field need to be improved.

In the paper, a novel 2-DOF (degree of freedom) plane translational parallel manipulator with passive universal joints and three legs is presented. Firstly, the 2-DOF translational parallel manipulator which has the spatial structure and high bearing capacity in the direction perpendicular to the kinematics plane is described. Then, the kinematics analysis of the 2-DOF parallel manipulator, which include inverse and forward solutions, are studied in detail, and the Jacobian matrix of the parallel manipulator is also derived based on it. Lastly, to improve the stability and bearing capacity further, the symmetric mechanisms with four legs and passive universal joints are constructed by adding a leg in parallel. The proposed 2-DOF parallel manipulator not only has the simple structure, but high stiffness especially in the direction perpendicular to kinematics plane for its spatial arrangment and passive universal joints.