Article

Assay optimization and kinetic profile of the human and the rabbit isoforms of 11beta-HSD1.

Pfizer Global Research and Development, La Jolla Laboratories, 10628 Science Center Drive, San Diego, CA 92121, USA.
Biochemical and Biophysical Research Communications (impact factor: 2.48). 07/2007; 357(2):561-6. DOI:10.1016/j.bbrc.2007.04.005 pp.561-6
Source: PubMed

ABSTRACT Assay conditions for the 11beta-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K(i)a as well as the apparent Michaelis-Menten constants K(m)a, K(m)b, k(cat)a, and k(cat)b for NADPH and cortisone, have been determined to be 147.5 microM, 14.4 microM, 43.8 nM, 0.21 min(-1), and 0.27 min(-1), respectively, for the human enzyme and 41.1 microM, 3.1 microM, 161.7 nM, 0.49 min(-1), and 0.52min(-1), respectively, for the rabbit enzyme.

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Keywords

11beta-hydroxysteroid dehydrogenase
 
apparent Michaelis-Menten constants K(m)a
 
Assay conditions
 
bind
 
binding
 
cortisone
 
enzyme catalytic site
 
enzymes
 
equilibrium dissociation constant
 
human enzyme
 
Kinetic analyses
 
ordered sequential bi-bi mechanism
 
rabbit enzyme isoforms