Abstract
A crucial enzyme in the pathway for protein degradation in Escherichia coli is protease La, an ATP-hydrolyzing protease encoded by the lon gene. This enzyme degrades various proteins to small polypeptides containing 10-20 amino acid residues. To learn more about its energy requirement, we determined the number of ATP molecules hydrolyzed by the purified protease for each peptide bond cleaved. The enzyme hydrolyzed about 2 molecules of ATP for each new amino group generated with casein, bovine serum albumin, glucagon, or guanidinated casein as substrates, even though these proteins differ up to 20-fold in size and 3-4 fold in rates of hydrolysis of peptide bonds. Similar values for the stoichiometry (from 1.9 to 2.4) were obtained using fluorescamine or 2,4,6-trinitrobenzene sulfonic acid to estimate the appearance of new amino groups. These values appeared lower at 1 mM than at 10 mM Mg2+. The coupling between ATP and peptide bond hydrolysis appeared very tight. However, when the protease was assayed under suboptimal conditions (e.g. at lower pH or with ADP present), many more ATP molecules (from 3.5 to 12) were consumed per peptide bond cleaved. Our data would indicate that the early steps in protein degradation consume almost as much energy (2 ATPs for each cleavage) as does the formation of peptide bonds during protein synthesis.
Highlights
A crucial enzyme in the pathway for protein degra- which reduce the contentof protease La(18,19),decrease the dation in Eecherichia coli is protease La, an ATP- cell’s capacity to degrade abnormalproteins (16-18, 20)
Inhibition of the ATPase activity by nonmetabolizable ATP analogs or by vanadate (6, 8, 9) causes a proportionate reduction in protein breakdown, while inhibibumin, glucagon, or guanidinated casein as substrates, tion of proteolytic activity with diisopropyl fluorophosphate even though these proteins differ up to 20-fold in size reduces its ATPase activity (8).Probably the strongest eviand 3-4-fold in rates of hydrolysis of peptide bonds. dence for atight coupling between these activities is that
We have proposed a cyclical multistep mechanism (23, 24, 38) in which ATP binding first occurs, followedby peptide bond cleavage and ATPhydrolysis
Summary
Protease La was purified, as described by Waxman and Goldberg f [ IO PROTEIN STIMULATED ATPase. ATP, ADP, 0-methylisourea, fluorescamine, leucine, EDTA, and a-casein were obtained from Sigma. 1 mM ATP, 10 mM M$+,50 mM HEPES (pH 7.9), and 10 pg of the protein substrate in a total volume of 200 pl. The assay tubes were incubated at 37 "C for varying periods of time duringwhich the rates of proteolysis were linear (Fig. 1).To test if the stoichiometry. 15 30 45 60 for ATP-dependent proteolysis depended on the substrate degraded, we compared the degradation of a-casein,a-caseinin which free amino groups were blocked by guanidination with 0-methylisourea (25),glucagon, and BSA denatured by reduction and carboxyamidomethylation (26). The ATPase activity was assayed at the same time in the same buffer, generally ATP was present at 0.5 mM to reduce the blank values in the determination of inorganic phosphate.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
