PMID:12962509

Citation	Bruckner, RC, Gunyuzlu, PL and Stein, RL (2003) Coupled kinetics of ATP and peptide hydrolysis by Escherichia coli FtsH protease. Biochemistry 42:10843-52
Abstract	FtsH from Escherichia coli is an ATP- and Zn(2+)-dependent integral membrane protease that is involved in the degradation of regulatory proteins such as sigma(32) and uncomplexed subunits of membrane protein complexes such as secY of the protein translocase. We describe a protocol for solubilizing the recombinant enzyme from inclusion bodies and its subsequent refolding and purification to near homogeneity. This is a high-yield protocol and produces in excess of 20 mg of purified FtsH per liter of E. coli culture. We found that refolded FtsH has biochemical properties similar to detergent extracted overexpressed protein described previously. FtsH forms a large complex with an apparent mass of 1200 kDa as determined by gel filtration. Both ATPase and protease activities are coincident with this large complex; smaller forms of FtsH do not exhibit either activity. While FtsH-catalyzed hydrolysis of ATP can occur in the absence of protein substrate (k(c) = 22 min(-1); K(m) = 23 microM), proteolysis shows an absolute dependence on nucleoside-5'-triphosphates, including ATP, CTP, and various analogues. In the presence of 5 mM ATP, FtsH catalyzes the hydrolysis of sigma(32) with the following observed kinetic parameters: k(c) = 0.18 min(-1) and K(m) = 8.5 microM. Significantly, this reaction is processive and generates no intermediate species, but rather, approximately 10 peptide products, all of MW <3 kDa. FtsH protease also efficiently hydrolyzes the peptide Phe-Gly-His-(NO)2Phe-Phe-Ala-Phe-OMe. Hydrolysis occurs exclusively at the (NO)2Phe-Phe bond (k(c) = 2.1 min(-1); K(m) = 12 microM), and like proteolysis, shows an absolute dependence on NTPs. We propose a mechanism for the coupled hydrolytic activities of FtsH toward ATP and peptide substrates that is consistent with a recently proposed structural model for FtsH.
Links	PubMed Online version:10.1021/bi034516h
Keywords	ATP-Dependent Proteases; Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Chromatography, Gel; Consensus Sequence; Endopeptidases/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Heat-Shock Proteins/chemistry; Heat-Shock Proteins/metabolism; Hydrolysis; Kinetics; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/isolation & purification; Membrane Proteins/metabolism; Models, Molecular; Oligopeptides/chemistry; Oligopeptides/metabolism; Protein Folding; Protein Structure, Tertiary; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sigma Factor/chemistry; Sigma Factor/metabolism; Substrate Specificity; Transcription Factors/chemistry; Transcription Factors/metabolism; Zinc/chemistry; Zinc/metabolism
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