Escherichia coli Lon, also known as protease La, is an oligomeric ATP-dependent protease, which functions to degrade damaged and certain short-lived regulatory proteins in the cell. To investigate the kinetic mechanism of E. coli Lon protease, we performed the first pre-steady-state kinetic characterization of the ATPase and peptidase activities of this enzyme. Using rapid quench-flow and fluorescence stopped-flow spectroscopy techniques, we demonstrated that ATP hydrolysis occurs before peptide cleavage, with the former reaction displaying a burst and the latter displaying a lag in product production. The detection of burst kinetics in ATP hydrolysis is indicative of a step after nucleotide hydrolysis being rate-limiting in ATPase turnover. At saturating substrate concentrations, the lag rate constant for peptide cleavage is comparable to the kcat of ATPase, indicating that two hydrolytic processes are coordinated during the first enzyme turnover. The involvement of subunit interaction during enzyme catalysis was detected as positive cooperativity in the binding and hydrolysis of substrates, as well as apparent asymmetry in the ATPase activity in Lon. When our data are taken together, they are consistent with a reaction model in which ATP hydrolysis is used to generate an active enzyme form that hydrolyzes peptide.

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.

Lon is an ATP-dependent serine protease which degrades damaged and certain regulatory proteins in vivo. Lon exists as a homo-oligomer and represents one of the simplest ATP dependent proteases because both the protease and ATPase domains are located within each monomeric subunit. Previous pre-steady state kinetic studies revealed functional non-equivalency in the ATPase activity of the enzyme [Vineyard, D., et al. (2005) Biochemistry 44, 1671. Both a high and low affinity ATPase site has been previously reported for Lon [Menon, A.S., & Goldberg, A.L. (1987) J Biol Chem 262, 14921-8]. Because of the differing affinities for ATP, we were able to monitor the activities of the sites separately and determine that they were non-interacting. The high affinity sites hydrolyze ATP very slowly (k obs =0.019 ± 0.002s −1 ), while the low affinity sites hydrolyze ATP quickly at a rate of 17.2 ± 0.09s −1 which is comparable to the previously observed burst rate. Although the high affinity sites hydrolyze ATP slowly they support multiple rounds of peptide hydrolysis, indicating that ATP and peptide hydrolysis are not stoichiometrically linked. However, ATP binding and hydrolysis at both the high and low affinity sites is necessary for optimal peptide cleavage and the stabilization of the conformational change associated with nucleotide binding.Lon is an ATP dependent serine protease functioning to degrade damaged and certain regulatory proteins in vivo (1-10). Lon belongs to the ATPases associated with a variety of cellular activities (AAA + ) superfamily whose members include ClpAP, ClpXP, ClpCP, and HslVU (11,12). They share a conserved Walker A (or P-loop) and Walker B motif which is associated with nucleotide binding and hydrolysis (13). Lon represents one of the simplest of the ATP dependent proteases because both the protease and ATPase domains are located within each monomeric subunit (14,15). Crystal structures of portions of the enzyme have been recently reported, and include an inactive mutant of the Lon protease domain (16)(17)(18). This structure shows Lon as a hexamer organized in a ring with a central cavity which is commonly found in other ATP dependent proteases (11,13,17). Although it is known that ATP modulates Lon's protease activity (4,5,7,19), mechanistic details concerning how the binding and hydrolysis of ATP is coordinated with peptide bond cleavage is not known. However, it has been shown that ATP binding and hydrolysis does not affect the oligomeric state of the enzyme (20,21).We have previously developed a continuous fluorescent peptidase assay to monitor the kinetics of peptide cleavage. As the inner filter effect of fluorescence interferes at high concentrations of 100% fluorescent peptide, we used S3, a 10% mixture of fluorescently labeled peptide with its non-fluorescent analog (S2) (22). No optical signal from the peptide is needed when monitoring ATPase activity so only the non-fluorescent analog (S2) is used to account for the *Corresponding author, email: Irene.lee@case.edu, NIH Pub...

The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the pi-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of shape and pi-electron density. In this report, we describe the synthesis and kinetic characterization of four novel nucleotide analogs, 5-cyanoindolyl-2'-deoxyriboside 5'-triphosphate (5-CyITP), 5-ethyleneindolyl-2'-deoxyriboside 5'-triphosphate (5-EyITP), 5-methylindolyl-2'-deoxyriboside 5'-triphosphate (5-MeITP), and 5-ethylindolyl-2'-deoxyriboside 5'-triphosphate (5-EtITP). Kinetic analyses indicate that the overall catalytic efficiencies of all four nucleotides are related to their base-stacking properties. In fact, the catalytic efficiency for nucleotide incorporation opposite an abasic site displays a parabolic trend in the overall pi-electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA approximately 100-fold worse than opposite an abasic site. These data indicate that selectivity for incorporation opposite damaged DNA can be achieved through optimization of the base-stacking properties of the incoming nucleotide.

Lon is an oligomeric serine protease whose proteolytic activity is mediated by ATP hydrolysis. Although each monomeric subunit has an identical sequence, Lon contains two types of ATPase sites that hydrolyze ATP at drastically different rates. The catalytic low-affinity sites display pre-steady-state burst kinetics and hydrolyze ATP prior to peptide cleavage. The high-affinity sites are able to hydrolyze ATP at a very slow rate. By utilizing the differing Kd's, the high-affinity site can be blocked with unlabeled nucleotide while the activity at the low-affinity site is monitored. Little kinetic data are available that describe microscopic events along the reaction pathway of Lon. In this study we utilize MANT-ATP, a fluorescent analogue of ATP, to monitor the rate constants for binding of ATP as well as the release of ADP from Escherichia coli Lon protease. All of the adenine nucleotides tested bound to Lon on the order of 10(5) M(-1) s(-1), and the previously proposed conformational change associated with nucleotide binding was also detected. On the basis of the data obtained in this study we propose that the rate of ADP release is slightly different for the two ATPase sites. As the model peptide substrate [S2; YRGITCSGRQK(Bz)] [Thomas-Wohlever, J., and Lee, I. (2002) Biochemistry 41, 9418-9425] or the protein substrate casein affects only the steady-state ATPase activity of the low-affinity sites, we propose that Lon adopts a different form after its first turnover as an ATP-dependent protease. Based on the obtained rate constants, a revised kinetic model is presented for ATPase activity in Lon protease in both the absence and presence of the model peptide substrate (S2).