Open AccessEnzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium
Author(s) -
MITAMURA Toshihide,
URABE Itaru,
OKADA Hirosuke
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb15221.x
Subject(s) - thermostability , bacillus megaterium , enzyme , isozyme , nad+ kinase , michaelis–menten kinetics , chemistry , dehydrogenase , biochemistry , escherichia coli , substrate (aquarium) , cofactor , enzyme assay , biology , bacteria , gene , genetics , ecology
Three glucose dehydrogenases (GlcDH) from Bacillus megaterium , GlcDH‐I, GlcDH‐II and GlcDH‐IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDK1 and pGDK2 contain two isozyme genes, gdhI and gdhII , respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH‐IWG3 is a variant of GlcDH‐I. GlcDH‐I and GlcDH‐II have similar pH/activity profiles and the profile for GlcDH‐IWG3 is identical to that of GlcDH‐I. The pH/stability profiles of these enzymes show that GlcDH‐IWG3 is the most stable enzyme in the acidic region, while GlcDH‐II is the most stable in the alkaline region, and GlcDH‐I is the most unstable throughout the entire pH range examined. As for thermostability, GlcDH‐II is the most resistant against heat inactivation at pH 6.5. The values of the first‐order rate constant for heat inactivation at 50°C are 0.27 min −1 , 0.05 min −1 and 0.11 min −1 for GlcDH‐I, GlcDH‐II and GlcDH‐IWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in K ia for NAD(P) and K a (the limiting Michaelis constant) for NAD; the values of the ratio of K ia for NAD and NADP are 11340 and 8.7 for GlcDH‐I, GlcDH‐II and GlcDH‐IWG3, respectively. GlcDH‐I and GlcDH‐IWG3 have very similar substrate specificities and GlcDH‐II has a slightly higher specificity for D‐glucose and 2‐deoxy‐D‐glucose than the others. The results are discussed on the basis of the amino acid substitutions between the enzymes.