Second derivative spectroscopy of enolase at high hydrostatic pressure: an approach to the study of macromolecular interactions.
Second derivative spectroscopy in the ultraviolet region of proteins has been used to study the polarity of the regions surrounding tyrosine residues. We show here that it can also be a tool to study the degree to which proteins associate and that it can be effectively combined with hydrostatic pressure in order to evaluate equilibrium dissociation constants and reaction volumes. Hydrostatic pressure causes yeast enolase to dissociate. Clear changes in the second derivative spectra of enolase were observed as pressure was increased. At enolase concentrations of about 20 microM, the midpoint of the transition is about 1800 bar. All aspects of the transition are reversible up to 2700 bar. It is likely that the transition observed is the result of enolase dimers dissociating into monomers. The second derivative spectra indicate that one or more tyrosine residues is in an unusually polar environment in the dimer, an environment that is less polar in the monomer. Three tyrosines (6, 11, 130) are near the dimer interface. Tyrosines 6 and 11 are pointing into the water-filled crevice between the subunits and are close to several immobilized waters. All three are close to a network of intersubunit salt bridges and hydrogen bonds. We believe that the average tyrosine polarity in the dimer reflects the exposure of these tyrosines to immobilized water and the fixed dipole of the salt bridge. The water in the crevice between the subunits should be more mobile in the monomer; the salt bridge does not exist in the monomer.(ABSTRACT TRUNCATED AT 250 WORDS)
Kornblatt
J A
JA
Department of Biology, Concordia University, Montreal, Quebec, Canada.
Kornblatt
M J
MJ
Hoa
G H
GH
D006874:Hydrostatic Pressure; D046911:Macromolecular Substances; D009113:Muramidase / Q000737:chemistry; D010751:Phosphopyruvate Hydratase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D015003:Yeasts / Q000201:enzymology
D046911:Macromolecular Substances; D014443:Tyrosine; D014364:Tryptophan; D009113:Muramidase; D010751:Phosphopyruvate Hydratase