Stabilization of creatinase from Pseudomonas putida by random mutagenesis.
Creatinase (creatine amidinohydrolase, EC 3.5.3.3) from Pseudomonas putida is a homodimer of 45 kDa subunit molecular mass, the three-dimensional structure of which is known at 1.9 A resolution. Three point mutants, A109V, V355M, and V182I, as well as one double mutant combining A109V and V355M, and the triple mutant with all three replacements, were compared with wild-type creatinase regarding their physical and enzymological properties. High-resolution crystal data for wild-type creatinase and the first two mutants suggest isomorphism at least for these three proteins (R. Huber, pers. comm.). Physicochemical measurements confirm this prediction, showing that the mutations have no effect either on the quaternary structure and gross conformation or the catalytic properties as compared to wild-type creatinase. The replacement of V182 (at the solvent-exposed end of the first helix of the C-terminal domain) does not cause significant differences in comparison with the wild-type enzyme. The other point mutations stabilize the first step in the biphasic denaturation transition without affecting the second one. In sum, the enhanced stability seems to reflect slight improvements in the local packing without creating new well-defined bonds. The increase in hydrophobicity generated by the introduction of additional methyl groups (A109V, V182I) must be compensated by minor readjustments of the global structure. Secondary or quaternary interactions are not affected. In going from single to double and triple mutants, to a first approximation, the increments of stabilization are additive.
Schumann
J
J
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Böhm
G
G
Schumacher
G
G
Rudolph
R
R
Jaenicke
R
R
D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D016296:Mutagenesis; D009604:Nitrosoguanidines / Q000494:pharmacology; D011489:Protein Denaturation; D016958:Pseudomonas putida / Q000201:enzymology*; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D014512:Ureohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006146:Guanidines; D046911:Macromolecular Substances; D009604:Nitrosoguanidines; D014508:Urea; D014512:Ureohydrolases; C019944:creatinase; D019791:Guanidine