Enzyme sequence and its relationship to hyperbaric stability of artificial and natural fish lactate dehydrogenases.
The cDNAs of lactate dehydrogenase b (LDH-b) from both deep-sea and shallow living fish species, Corphaenoides armatus and Gadus morhua respectively, have been isolated, sequenced and their encoded products overproduced as recombinant enzymes in E. coli. The proteins were characterised in terms of their kinetic and physical properties and their ability to withstand high pressures. Although the two proteins are very similar in terms of their primary structure, only 21 differences at the amino acid level exist between them, the enzyme from the deep-sea species has a significantly increased tolerance to pressure and a higher thermostability. It was possible to investigate whether the changes in the N-terminal or C-terminal regions played a greater role in barophilic adaptation by the construction of two chimeric enzymes by use of a common restriction site within the cDNAs. One of these hybrids was found to have even greater pressure stability than the recombinant enzyme from the deep-living fish species. It was possible to conclude that the major adaptive changes to pressure tolerance must be located in the N-terminal region of the protein. The types of changes that are found and their spatial location within the protein structure are discussed. An analysis of the kinetic parameters of the enzymes suggests that there is clearly a trade off between K(m) and k(cat) values, which likely reflects the necessity of the deep-sea enzyme to operate at low temperatures.
Brindley
Amanda A
AA
Department of Biosciences, University of Kent, Canterbury, Kent, United Kingdom. a.a.brindley@kent.ac.uk
Pickersgill
Richard W
RW
Partridge
Julian C
JC
Dunstan
David J
DJ
Hunt
David M
DM
Warren
Martin J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005399:Fishes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D045022:Lactate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011312:Pressure; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics
D045022:Lactate Dehydrogenases