A mutation study of catalytic residue Asp 52 in hen egg lysozyme.
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 pH 5.0 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 35 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.
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Y
Y
Hashimoto
K
K
Yamada
H
H
Motoshima
T
T
Omura
H
H
Yamada
T
T
Yasukochi
T
T
Miki
T
T
Ueda
T
T
Imoto
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D002645:Chickens / Q000235:genetics*; D018360:Crystallography, X-Ray; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D015398:Signal Transduction; D043324:beta-Fructofuranosidase
D004527:Egg Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; D009113:Muramidase; D043324:beta-Fructofuranosidase