Abstract
The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.
Highlights
La fromEscherichia colienhances its ability tohydro- teins, approximately two ATP molecules are hydrolyzed for lyze ATP and peptide bonds
Studies with model peptides (13) indicate that thisunusual bound, ATP is quickly hydrolyzed to ADP, which re- protease interacts with protein substratesat two distinct sites: mains noncovalently associated with protease Laeven one is the active site, which preferentially hydrolyzes hydrothrough repeated gel filtrations
Protein substrates stimulate 2-4-fold the the enzyme were not released by these substrates.Proteins that arenot degraded andoligopeptides that only bind to the catalytic sitedo not induce ADP release
Summary
MATERIALS ANDMETHODS affinity of protease Lafor this nucleotide analog. Protease La was purified as described earlier (13). The binding of ATP or ADP to protease La was studied by centrifugation of a ligand-enzyme mixture through Sephadex G-50, as described in our preceding paper (16). To study the release of bound ATP from protease La the enzyme["]ADP complex was isolated by centrifugation through Sephadex G-50columns (16).The eluted protease-ligand complex wasincubated with protein substrates for varying periods of time at 37 "C, and the reaction mixture was centrifuged again through Sephadex G-50 columns at 100 X g for 5 min. One possible mechanism for the protein-stimulated ATPase activity would be if ADP release were therate-limitingstepand if polypeptides enhanced release of ADP from theenzyme To test this possibility, the enzyme was incubated with [3H]ATP and theenzyme-nucleotide complex was isolated by centrifugation through Sephadex G-50 columns and incubated witha substrate (e.g. acasein) ora nondegraded protein (nativeBSA) a t 37 "C.
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