Biomaterial Database

Stopped-flow cryoenzymology: detection of catalytic intermediates not observable at ambient temperatures. 1983

H E Van Wart, and S H Lin

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.

UI	MeSH Term	Description	Entries
D007668	Kidney	Body organ that filters blood for the secretion of URINE and that regulates ion concentrations.	Kidneys
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D007931	Leucyl Aminopeptidase	A zinc containing enzyme of the hydrolase class that catalyzes the removal of the N-terminal amino acid from most L-peptides, particularly those with N-terminal leucine residues but not those with N-terminal lysine or arginine residues. This occurs in tissue cell cytosol, with high activity in the duodenum, liver, and kidney. The activity of this enzyme is commonly assayed using a leucine arylamide chromogenic substrate such as leucyl beta-naphthylamide.	Cytosol Aminopeptidase,Leucine Aminopeptidase,L-Leucylnaphthylamidase,Methoxyleucine Aminopeptidase,Peptidase S,Zinc-Manganese-Leucine Aminopeptidase,Aminopeptidase, Cytosol,Aminopeptidase, Leucine,Aminopeptidase, Leucyl,Aminopeptidase, Methoxyleucine,Aminopeptidase, Zinc-Manganese-Leucine,Zinc Manganese Leucine Aminopeptidase
D009991	Oscillometry	The measurement of frequency or oscillation changes.	Oscillometries
D003600	Cytosol	Intracellular fluid from the cytoplasm after removal of ORGANELLES and other insoluble cytoplasmic components.	Cytosols
D005615	Freezing	Liquids transforming into solids by the removal of heat.	Melting
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D013552	Swine	Any of various animals that constitute the family Suidae and comprise stout-bodied, short-legged omnivorous mammals with thick skin, usually covered with coarse bristles, a rather long mobile snout, and small tail. Included are the genera Babyrousa, Phacochoerus (wart hogs), and Sus, the latter containing the domestic pig (see SUS SCROFA).	Phacochoerus,Pigs,Suidae,Warthogs,Wart Hogs,Hog, Wart,Hogs, Wart,Wart Hog
D013696	Temperature	The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.	Temperatures