TIME-RESOLVED X-RAY-DIFFRACTION STUDIES OF ENZYMES UNDER CRYOCONDITIONS

Cryoenzymological techniques provide a means of initiating enzymatic reactions in crystals homogeneously and of prolonging the lifetimes of intermediate reaction steps. Catalytic intermediates may accumulate to sufficiently high population for X-ray crystal structure analysis by time-resolved monochromatic or Laue diffraction data collection using synchrotron radiation. Due to short exposure times, intermediates may be studied at moderately low temperatures. A combination of cryoenzymology and time-resolved crystallography has been applied to crystal structure analysis at high resolution of an acyl-enzyme intermediate of a productive reaction catalysed by porcine pancreatic elastase.