Chemical Modification and Site-Directed Mutagenesis

Group-specific chemical modification remains a useful method for studies of structure-function relationships in protein molecules, although unambiguous identification of essential amino acid residues and elucidation of their function are nowadays accomplished mainly by X-ray crystallography and site-directed mutagenesis. Chemical modifications 

Morino and Snell32 reduced holotryptophanase with NaBIL and isolated -pyridoxyl-lysine from the hydrolyzed protein. Subsequent isolation of the pyridoxyl-containing peptide50 and determination of its position in the total amino acid sequence of the enzyme led to identification of Lys-270, which forms a Schiff base with PLP. 

The adjacent residue, Lys-269 was replaced by arginine by site-directed mutagenesis.51 The mutant enzyme exhibited the kcat value about 90% lower than that of the wild-type enzyme, and the kcatl Km value about 98% lower. The reaction of the mutant enzyme with substrates and L-alanine was not accompanied by accumulation of the quinonoid intermediate. Phillips et a/.5l) suggested that Lys-269 plays a significant role in the electrostatic interactions or conformational changes obligatory to the formation and breakdown of the quinonoid intermediate, although it is not a catalytically essential residue. 

Nihira, Toraya and Fukui53 found that irradiation of holotryptophanase solutions with visible light led to rapid, pH-dependent loss of enzymatic activity. The apoenzyme was not inactivated under the same conditions therefore, the inactivation was PLP-sensitized. L-Tryptophan and l-alanine markedly decreased the rate of photoinactivation. From a plot of the inactivation rate versus pH, the pKa was found to be 7.2, which is close to the pKa of the imidazole group. Photoinactivation was accompanied by destruction of one histidine residue per subunit. No concomitant loss of tryptophan, tyrosine or methionine was detected, and it was suggested that tryptophanase contains an essential histidine residue near the active site. 

The presence of an essential tyrosine residue near the active site was suggested on the basis of experiments with tetranitromethane.54 Treatment of apotryptophanase with this reagent caused almost complete loss of catalytic activity, a great reduction of affinity for PLP and modification of about one tyrosine residue. The modified enzyme was unable to form the quinonoid intermediate with L-tryptophan or L-alanine.55 PLP protected the apoenzyme from inactivation only in the presence of activating cations (K+, NH4+, Rb+). It was shown that inactivation by tetranitromethane was not caused by oxidation of SH-groups, but partial modification of methionine (0.8 residue) was detected and might also be responsible for inactivation. It is worthy of note that modification of tryptophanase with chloramine T indicated that some methionine residues may be important for maintaining the catalytically active conformation of the enzyme.56  

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Anstead et al. [18] conducted a comprehensive review of SARs between steroidal ligands and ERbinding affinity. These were deduced from indirect studies of the ER involving chemical modification and site-directed mutagenesis. Through this research, the importance of specific residues for binding to the ER has been deduced. 

Comparison of Imidazole-containing Antibodies Produced by Chemical Modification and Site-directed Mutagenesis... 

Hartlieb, J., Rilterjans, H. (2001). Insights into the reaction mechanism of the diisopropyl-fluorophosphatase Jrom Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis. Biochim. Biophys. Acta 1546 312-24. 

Active-site modifications. Chemical modification and site-directed mutagenesis experiments suggest that Glu-270 is essential for cataly-... 

Chemical modifications and site-directed mutagenesis identihcation of catalytic and/or binding residues involved ... 

Ishii Y. Saito Y, Fujimura T, Nc uchi Y, Sasaki H, Niwa M, Shimomura K. High-level production, chemical modification and site-directed mutagenesis of cephalosporin C acylase fifom Pseudomonas sp. N176. Eur J Biochem 1995 230 77,3-778. 

Nobbs TJ. Ishii Y, Fujimura T, Saito Y. Niwa M. Chemical modification and site-directed mutagenesis of tyrosine residues in cephalosporin C acylase from Pseudomonas strain N176. ] Ferment Bioeng 1994 77 604-609. 

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