Proteolytic activity of the ATP-dependent protease HslVU can be uncoupled from ATP hydrolysis.
HslVU is a new Escherichia coli ATP-dependent protease composed of two multimeric complexes: the HslU ATPase and the HslV peptidase. Prior studies indicated that HslVU requires ATP hydrolysis for the cleavage of peptides and proteins. We show here that ATP concentrations that activate hydrolysis of benzyloxycarbonyl-Gly-Gly-Leu-7-amido-4-methylcoumarin are 50-100 fold lower than those necessary for degradation of proteins (e.g. casein). Also, the nonhydrolyzable analogs of ATP, 5'-adenylyl beta, gamma-imidodiphosphate (AMP-PNP) and adenosine 5'-(alpha, beta-methylene)triphosphate, can support peptide hydrolysis, but only after an initial time lag not seen with ATP. This delay decreased at higher temperatures and with higher HslU or HslV concentrations and was eliminated by preincubation of HslU and HslV together. Thus, ATP hydrolysis accelerates the association of HslU and HslV, which occurs slowly with the nonhydrolyzable analog. The addition of KCl stimulated 4-6-fold the peptidase activity with AMP-PNP present and eliminated the time lag, but KCl had no stimulatory effect with ATP. NH4+ and Cs+ had similar effects as K+, but Na+ and Li+ were ineffective. AMP-PNP by itself supported hydrolysis of casein and other polypeptides only 20% as well as ATP, but in the presence of K+, Cs+, or NH4+, AMP-PNP activated casein degradation even better than ATP, although it was not hydrolyzed. In addition, MgCl2, MnCl2, and CaCl2 allowed some peptidase and caseinase activity in the absence of any nucleotide. However, Mn2+ and Ca2+, unlike Mg2+, abolished ATP hydrolysis and prevented further activation by ATP or AMP-PNP. These findings indicate that ATP binding to a high affinity site triggers the formation of an active state capable of peptide cleavage, although ATP hydrolysis facilitates this process. Rapid degradation of proteins requires a distinct state of the enzyme, which is normally reached through ATP hydrolysis at low affinity sites. However, AMP-PNP binding together with K+ can induce a form of HslVU that degrades proteins without energy consumption.
Huang
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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Goldberg
A L
AL
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D002413:Cations, Divalent; D002414:Cations, Monovalent; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006360:Heat-Shock Proteins; D006868:Hydrolysis; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012697:Serine Endopeptidases; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D002414:Cations, Monovalent; D006360:Heat-Shock Proteins; D009711:Nucleotides; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D000251:Adenosine Triphosphatases; D011188:Potassium