Elucidation of the chemical nature of the steady-state intermediates in the mechanism of carboxypeptidase A

A Galdes, D S Auld, B L Vallee
Biochemistry 1986 February 11, 25 (3): 646-51
Cryospectrokinetic studies of zinc and cobalt carboxypeptidase A disclosed two intermediates in the hydrolysis of both peptides and depsipeptides and furnished all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2---E + P [Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., & Vallee, B. L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045]. Since the ES2 intermediate is the predominate enzyme species present at steady state, its chemical nature is deducible from subzero chemical quench studies done after steady state is established. Extrapolation of the product concentration to zero time, [P0], measures the concentration of the enzyme species in which bond cleavage has occurred. For peptides, the [P0]values are zero, indicating that no product is generated prior to turnover and therefore the ES2 intermediate involves a complex between enzyme and intact peptide substrate. For depsipeptides, [P0] values are 1 mol of produce per mole of enzyme over the entire temperature range -20 to -50 degrees C, indicating cleavage of the ester bond occurs prior to the rate-limiting step so that ES2 is more properly denoted by EP1P2, where P1 and P2 are the substrates for the reverse reaction. The rate-limiting step for depsipeptides thus involves release of the products which may occur directly or through a mandatory conformational change followed by rapid product release.

Full Text Links

Find Full Text Links for this Article


You are not logged in. Sign Up or Log In to join the discussion.

Trending Papers

Remove bar
Read by QxMD icon Read

Save your favorite articles in one place with a free QxMD account.


Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"