Competitive Inhibition of Enzyme Activity by Urea*

It is widely accepted that urea and guanidine act as protein denaturants by breaking intramolecular hydrogen bonds (1). Loss of catalytic activity in the presence of urea is thought to occur by elimination of bonds contributing to the tertiary structure of enzyme molecules. Subsequent restoration of structural and catalytic properties by removal of the denaturant, “reversible denaturation,” is usually interpreted as evidence of the reformation of the ruptured hydrogen bonds. Although there exists a large literature describing denaturation of enzymes by urea, there have been few kinetic analyses of the reversible inhibition of enzyme activity at relatively low levels of urea. Although ribonuclease activity appears to be unaffected in 8 M urea (2)) it has been shown that the ribonuclease molecule is actually extensively unfolded in such solutions, but the process is reversed by its substrate, ribonucleic acid (or other polyanions), so that there is no apparent influence on the enzymatic activity of the molecule (3). Pepsin (4) and carboxypeptidase (5) have been shown to retain their activity at relatively high urea concentrations. Reversible inactivation of trypsin (6), lysozyme (7), invertase (8), Cypridina luciferase (9), and phosphoglucomutase (10) by urea has been reported, whereas chymotrypsin (6) and aldolase (11) have been seen only to undergo irreversible inactivation in urea solution. Hill, Schwartz, and Smith (12) have studied the effect of relatively low concentrations of urea on the activity of papain; their data indicate that, with cu-benzoyl-L-argininamide as substrate, urea competitively inhibited papain. The present studies have demonstrated that in the presence of low concentrations of urea, milk xanthine oxidase exhibits an immediate inhibition which is readily reversed by dilution. Kinetic analysis revealed that urea serves as a formally competitive inhibitor of this enzyme. Twenty-one diverse enzymes have been similarly tested for their susceptibility to inhibition by urea and the nature of the inhibition characterized in each case. The results have revealed interesting differences in patterns of urea inhibition and are considered to bear on the mechanism of formation of the enzymesubstrate complex in each case. The competitive inhibition of enzymes by urea has been studied under different conditions in an effort further to understand the significance of the inhibition.