Low-temperature stopped-flow rapid-scanning spectroscopy: performance tests and use of aqueous salt cryosolvents.
An ultimate goal of enzymology is to determine the structure of intermediates in catalysis in solution. Metalloenzymes provide a unique opportunity in this regard because the metal atom is critical to catalysis. In the case of zinc enzymes, replacement of the zinc by the chromophoric cobalt atom can result in the identification of intermediates in catalysis. This is most easily accomplished by examining the enzyme-catalyzed reaction under rapid-mixing and rapid-scanning subzero conditions. Aqueous salt solutions are particularly useful as cryosolvents whenever hydrophobic forces are important for substrate binding. Rapid-freeze or chemical quenching of these metallointermediates can allow further structural characterization by such approaches as EPR spectroscopy and X-ray absorption fine structure. Such a combination of approaches can bridge the information gained by examining only the structure of the enzyme in the crystalline state by X-ray diffraction and that obtained by examining only steady-state enzymatic activities.
Auld
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Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
D000595:Amino Acid Sequence; D002510:Cephalosporinase / Q000737:chemistry; D003080:Cold Temperature; D004798:Enzymes / Q000737:chemistry*; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D012997:Solvents; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods*; D013379:Substrate Specificity
D004798:Enzymes; D008667:Metalloproteins; D008670:Metals; D009842:Oligopeptides; D012997:Solvents; D002510:Cephalosporinase