Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida.

Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) [Schwede, T.F., Rétey, J., Schulz, G.E. (1999) Biochemistry, 38, 5355-5361]. To evaluate the importance of several amino-acid residues at the active site for substrate binding and catalysis, we mutated the following amino-acid codons in the HAL gene: R283, Y53, Y280, E414, Q277, F329, N195 and H83. Kinetic measurements with the overexpressed mutants showed that all mutations resulted in a decrease of catalytic activity. The mutants R283I, R283K and N195A were approximately 1640, 20 and 1000 times less active, respectively, compared to the single mutant C273A, into which all mutations were introduced. Mutants Y280F, F329A and Q277A exhibited approximately 55, 100 and 125 times lower activity, respectively. The greatest loss of activity shown was in the HAL mutants Y53F, E414Q, H83L and E414A, the last being more than 20 900-fold less active than the single mutant C273A, while H83L was 18 000-fold less active than mutant C273A. We propose that the carboxylate group of E414 plays an important role as a base in catalysis. To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with l-cysteine and dioxygen at pH 10.5 as an indicator. All mutants, except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group. The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in HAL mutant F329A also. For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were performed.

Quality of life in chronic heart failure: cilazapril and captopril versus placebo. Cilazapril-Captopril Multicentre Group.

OBJECTIVE: To measure quality of life (QOL) in patients with mild to moderate heart failure treated with angiotensin converting enzyme (ACE) inhibitors cilazapril or captopril. DESIGN: Randomised, double blind, placebo controlled, parallel groups trial. SUBJECTS: 367 patients with New York Heart Association (NYHA) heart failure class II (62%), III (36%) or IV (1%). METHODS: Patients were randomised to receive cilazapril 1 mg daily (n = 191) or captopril 25 mg three times daily (n = 90) for 24 weeks, or placebo for 12 weeks followed by cilazapril 1 mg daily for a further 12 weeks (n = 86). If patients had not responded after four weeks cilazapril was increased to 2.5 mg daily and captopril to 50 mg three times daily. QOL was assessed at baseline, 12, and 24 weeks using the sickness impact profile (SIP), the profile of mood states (POMS), the Mahler index of dyspnoea-fatigue, and a health status index (HSI). RESULTS: The physical dimension of the SIP averaged 7 units at baseline and improved after 12 weeks by 2.24 units in the cilazapril group, 2.38 units in the captopril group, and 1.51 units in the placebo group. The difference between drug and placebo was therefore 0.73 units (95% CI -0.86 to 2.32) for cilazapril, and 0.87 units (95% CI -0.96 to 2.70) for captopril, with small non-significant effect sizes (a statistical method for estimating the importance of a treatment related change) of 0.12 and 0.14. Similar results were observed for the total POMS and HSI scores. Although QOL improved more on the ACE inhibitors than on placebo, the effect sizes were not significant (< or = 0.26). CONCLUSIONS: Improvements in QOL in mild to moderate heart failure were small when treated with cilazapril or captopril compared with placebo.