tailieunhanh - Báo cáo Y học: Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida

Institute for Organic Chemistry, University of Karlsruhe, Germany; 2Institute for Organic Chemistry, Budapest University of Technology and Economics, Hungary Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC ) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-i´ midazol-4-one (MIO) [Schwede, ., Retey, J., Schulz, . (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. | Eur. J. Biochem. 268 6011-6019 2001 FEBS 2001 Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida Daamar Rother1. László PóDDe2 Sandra Vierautz1 Biraid Lanaer1 and Janos Retev1 1 Institute for Organic Chemistry University of Karlsruhe Germany 2Institute for Organic Chemistry Budapest University of Technology and Economics Hungary Elucidation of the 3D structure of histidine ammonia-lyase HAL EC from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3 5-dihydro-5-methylidene-4H-i-midazol-4-one MIO Schwede . Retey J. Schulz . 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 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 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 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 .

• Báo cáo khoa học
• Report medicine
• traditional medicine
• scientific reports
• Crystal structure
• medical analysis
• biochemical studies
• Zizyphus mauritiana Lamk
• Biochemical constitutes
• Ber selection 1
• Studies on the pattern
• Pattern of changes biochemical constitutes
• Studies on Tikka disease
• Tikka disease
• Sowing dates
• Biochemical parameters of groundnut
• Biochemical parameters
• Studies on biochemical changes
• Biochemical changes
• Cell wall degrading enzymes
• Papaya fruit inoculated
• Colletotrichum demetium
• Vindhyan zone
• Haemato biochemical profile
• Haemato biochemical profile of goats
• Goats in Vindhyan region
• Biochemical variables
• Diaphragmatic hernia
• Various NSAIDS protocols affected
• Buffaloes treated
• Biochemical composition
• Reducing and Non reducing sugar
• Non reducing sugar
• Biochemical composition of various tomato
• Various tomato (Solanum lycopersicum L )
• environmental medicine
• Hepatobiliary disorders
• Serum biochemistry
• Hemato biochemical studies
• Hepatobiliary disorders in dogs
• Elevated total leucocyte
• Agricultural science
• Physiological and biochemical studies
• Wheat plants by application
• Amino acids and yeast extract
• Physio chemical
• Water relations
• Lucknow 49
• Ascarbic acid
• Different cultivars
• Guava under eastern
• Biochemical studies on adverse health
• Adverse health effects of arsenic
• Chromium in buffaloes
• Level of arsenic and chromium
• Heavy metals in buffaloes
• PyometraCEH complex
• Haemato biochemical evaluation
• Bitches affected
• Cystic endometrial hyperplasia pyometra complex
• Haemato biochemical studies
• Sloth bears
• HematoBiochemical changes
• Biochemical values
• Hemato biochemical changes
• Xylazine Zolazepam
• Sahiwal cows
• Blood metabolites
• Complete lactation in Sahiwal cows
• breast cancer
• medicine
• Hematopoietic
• medical knowledge