Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

[Phloroglucinol: safe and effective for the prevention of bladder spasm after TURP].

OBJECTIVE: To evaluate the efficacy of phloroglucinol in preventing bladder spasm after transurethral resection of the prostate (TURP). METHODS: Using the random sampling method, we assigned 74 cases of TURP into a treatment group (n = 39), given 80 mg phloroglucinol every day for 3 days, and a control group (n = 35), left untreated. Then we observed the frequency, duration and pain of bladder spasm within the 3 days and compared them between the two groups. RESULTS: The mean frequency, duration and pain visual analogue score of bladder spasm were (4.3 +/- 1.2) times, (7.2 +/- 2.1) min and 3.2 +/- 1.6 respectively in the treatment group, as compared with (7.5 +/- 2.4) times, (15.6 +/- 6.8) min and 4.7 +/- 2.3 in the control, with statistically significant differences between the two groups (P < 0.05). And no obvious adverse reactions were found in the treatment group. CONCLUSION: Phloroglucinol is safe and effective for the prevention and treatment of bladder spasm following TURP.