Temperature adaptation in Gillichthys (Teleost: Gobiidae) A(4)-lactate dehydrogenases: identical primary structures...

Alternative conformations of proteins underlie a variety of biological phenomena, from prion proteins that cause spongiform encephalopathies to membrane channel proteins whose conformational changes admit or exclude specific ions. In this paper, we argue that conformational differences within globular 'housekeeping' enzymes may allow rapid adaptation to novel environments. Muscle-type lactate dehydrogenases (A(4)-LDHs) from the gobies Gillichthys seta and G. mirabilis have identical amino acid sequences but show potentially adaptive differences in substrate affinity (apparent Michaelis constants for pyruvate, K(m)(PYR)) as well as differences in thermal stability. We examined the A(4)-LDH of each species using fluorescence spectroscopy, near- and far-ultraviolet circular dichroism (CD) spectroscopy and hydrogen/deuterium exchange (H/D) Fourier-transform infrared spectroscopy to determine whether structural differences were apparent, the extent to which structural differences could be related to differences in conformational flexibility and whether specific changes in secondary or tertiary structure could be defined. The fluorescence spectra and far-ultraviolet CD spectra of the A(4)-LDH from the two species were indistinguishable, suggesting that the two conformations are very similar in secondary and tertiary structure. Apparent melting temperatures (T(m)) followed by fluorescence and CD spectroscopy confirmed that the G. mirabilis A(4)-LDH is more thermally stable than the G. seta form. H/D exchange kinetics of Gillichthys A(4)-LDH was described using double-exponential regression; at 20 degrees C, G. seta A(4)-LDH has a higher exchange constant, indicating a more flexible and open structure. At 40 degrees C, the difference in H/D exchange constants disappears. Second-derivative analysis of H/D exchange infrared spectra indicates that alpha-helical, but not beta-sheet structure, differs in conformational flexibility between the two forms. Second-derivative ultraviolet spectra indicate that at least one of the five tyrosyl residues in the Gillichthys LDH-A monomer is located in a more hydrophobic environment in the G. mirabilis form. Homology models of A(4)-LDH indicate that Tyr246 is the most likely candidate to experience a modified environment because it is involved in subunit contacts within the homotetramer and sits in a hinge between a static alpha-helix and one involved in catalytic conformational changes. Subtle differences in conformation around this residue probably play a role both in altered flexibility and in the potentially adaptive differences in kinetics between the two A(4)-LDH forms.