Stopped-flow cryoenzymology: detection of catalytic intermediates not observable at ambient temperatures

Stopped-flow cryoenzymology has been used to study the reaction of porcine kidney cytosol leucine aminopeptidase [alpha-aminoacyl-peptide hydrolase(cytosol), EC 3.4.11.1] with L-leucylglycyldansyl hydrazide in 50% (vol/vol) methanol buffer over the -40 to 23 degrees C temperature range. Resonance energy transfer between tryptophan residues of the enzyme E and the dansyl group of the substrate S has been used to detect the formation and interconversion of reaction intermediates (E X S)i. Above 0 degrees C, a single intermediate E X S is formed and decays by first-order kinetics to products. However, at temperatures below -20 degrees C, a new intermediate (E X S)' is observed immediately after mixing, which relaxes to E X S within 100 msec. Because the detection of this new intermediate would not have been possible at ambient temperatures, this illustrates the value of stopped-flow cryoenzymology for studies of catalytic pathways.