We have performed random mutagenesis coupled with selection to isolate muta
nt enzymes with high catalytic activities at low temperature from thermophi
lic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermu
s thermophilus, Five cold-adapted mutant IPMDHs with single-amino-acid subs
titutions were obtained and analyzed. Kinetic analysis revealed that there
are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(
cat)) and K-m-improved (improved in k(cat)/K-m) types. To determine the mec
hanisms of cold adaptation of these mutants, thermodynamic parameters were
estimated and compared with those of the Escherichia coli wild-type IPMDH,
The DeltaG(m) values for Michaelis intermediate formation of the k(cat)-imp
roved-type enzymes were larger than that of the T. thermophilus wild-type I
PMDH and similar to that of the E. coli wild-type IPMDH. The DeltaG(m) valu
es of K-m-improved-type enzymes were smaller than that of the T. thermophil
us wild-type IPMDH, Fitting of NAD(+) binding was improved in the K-m-impro
ved-type enzymes. The two types of cold-adapted mutants employed one of the
two strategies of E. coli wild-type IPMDH: relative destabilization of the
Michaelis complex in k(cat)-improved-type, and destabilization of the rate
-limiting step in K-m-improved type mutants, Some cold-adapted mutant IPMDH
s retained thermostability similar to that of the T. thermophilus wild-type
IPMDH.