caa a22 4500 397555946 CHVBK 20180308164802.0 cr unu---uuuuu 161202e199601 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021199 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021199 A Mutation Study of Catalytic Residue Asp 52 in Hen Egg Lysozyme [Elektronische Daten] [Yoshio Hashimoto, Keiko Yamada, Hiroyuki Motoshima, Tadahiro Omura, Hidenori Yamada, Takanori Yasukochi, Takeyoshi Miki, Tadashi Ueda, Taiji Imoto] We constructed a system for the expression and secretion of mature hen lysozyme by yeast using an intermediate "secretion-signal cassette” vector, pKP1700, containing the yeast invertase signal sequence and an expression vector, pAM82, for secretion and maturation of the enzyme. Using this system, mutants of hen lysozyme were produced and the catalytic mechanism in hen lysozyme was definitely confirmed. The hydrolytic activity of D52A as to substrate (NAG)6 at pH5.0 was obviously decreased to one-four hundredth of that of the wild type. The acidic limb of the pH-activity profile observed for the wild-type was not observed for D52A, and the pKa of Glu 36 on the alkaline limb was seen for both enzymes. Moreover, no structural change was detected on X-ray analysis of D52A. Therefore, we confirmed that dissociated Asp 52 assists catalysis by producing an electrostatic field and by stabilizing the oxocarbonium ion intermediate in the dissociated form. © by the Journal of Biochemistry Regular Papers nationallicence catalytic mechanism nationallicence expression system nationallicence hen lysozyme nationallicence mutagenesis nationallicence X-ray analysis nationallicence Hashimoto Yoshio Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Yamada Keiko Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Motoshima Hiroyuki Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Omura Tadahiro Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Yamada Hidenori Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Yasukochi Takanori Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Miki Takeyoshi Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Ueda Tadashi Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut Imoto Taiji Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut The Journal of Biochemistry Oxford University Press 119/1(1996-01), 145-150 0021-924X 119:1<145 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021199 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021199 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hashimoto Yoshio Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Yamada Keiko Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Motoshima Hiroyuki Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Omura Tadahiro Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Yamada Hidenori Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Yasukochi Takanori Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Miki Takeyoshi Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Ueda Tadashi Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 700 1- Imoto Taiji Graduate School of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812-82 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/1(1996-01), 145-150 0021-924X 119:1<145 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford