Consequences of altered isoprenylation targets on a-factor export and bioactivity.

Cysteine-containing amino acid sequences (CAAX,CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any aminoacid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl)isoprenoids to peptides and proteins by specific prenyltransferases. Althoughmuch work has centered on the enzymatic mechanisms of these enzymes, thebiological consequences of the differential isoprenylation they catalyze remainto be elucidated. Farnesylation of the a-factor mating pheromone ofSaccharomyces cerevisiae is a known prerequisite for its biological activity andits secretion through a pathway utilizing the yeast STE6 protein, a homolog ofthe mammalian multidrug resistance (MDR) P-glycoprotein. We generated specificmutations in the a-factor gene to encode isoprenylation targets forgeranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in placeof the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factorscontaining these modified prenylation sites were successfully exported by aSTE6-dependent mechanism. Furthermore, these peptides, as well as syntheticgeranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons ofsynthetic and biosynthetic pheromones suggest that, in vivo, a peptide substratecontaining the geranylgeranylation target CVIL can be both farnesylated andgeranylgeranylated. These results clearly demonstrate that in vivo (i) differentprenyltransferases may recognize the same substrate; (ii) both farnesylated andgeranylgeranylated a-factor peptides are substrates for export via STE6, aMDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones areboth biologically active.     

Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity.

We have introduced a variety of amino acid substitutions into carboxyl-terminal CA1A2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic [Val12]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the A1 position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (approximately 50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.     

Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease.

The KEX2-encoded endoprotease was overproduced in yeast several hundred-fold and further purified to achieve a 10,000-fold enrichment in specific activity. The enzyme was (i) membrane-bound, but solubilized by detergents; (ii) able to cleave peptide substrates at both Lys-Arg and Arg-Arg sites; (iii) inhibited by EDTA and EGTA (but not o-phenanthroline), but fully reactivated by Ca2+; (iv) unaffected by 5-10 mM phenylmethylsulfonyl fluoride, N alpha-(ptosyl)lysine chloromethyl ketone, or L-1-tosylamido-2-phenylethyl chloromethyl ketone, but inactivated by 1-2 microM Ala-Lys-Arg-chloromethyl ketone; (v) labeled specifically by 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone; and (vi) resistant to trans-epoxysuccinate compounds (which inactivate thiol proteases), but inactivated by diisopropyl fluorophosphate (a diagnostic serine protease inhibitor). Mutant enzyme molecules lacking as many as 200 C-terminal residues still retained Ca2+-dependent protease activity and were labeled by 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone.     

Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage

Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated. The enzymes that mediate these activities, collectively referred to as CAAX processing components, have been identified genetically in Saccharomyces cerevisiae. Whereas the Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequence analysis predicts that the other CAAX processing components, the Rce1p and Ste24p proteases and the Ste14p methyltransferase, contain multiple membrane spans. To determine the intracellular site(s) at which CAAX processing occurs, we have examined the localization of the CAAX proteases Rce1p and Ste24p by subcellular fractionation and indirect immunofluorescence. We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane. In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of two cleavage steps that remove the amino-terminal extension from the a-factor precursor, suggesting that the first amino-terminal processing step of a-factor maturation also occurs at the ER membrane. The ER localization of Ste24p is consistent with the presence of a carboxyl-terminal dilysine ER retrieval motif, although we find that mutation of this motif does not result in mislocalization of Ste24p. Because the ER is not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routing of CAAX proteins from the ER membrane to other cellular sites.     

The endothelin-converting enzyme from human umbilical vein is a membrane-bound metalloprotease similar to that from bovine aortic endothelial cells.

A phosphoramidon-sensitive, membrane-bound metalloprotease that cleaves big endothelin 1 (big-ET-1) to ET-1 was obtained from human umbilical vein endothelial cells and also from bovine aortic endothelial cells by isolation of plasma-membrane vesicles free of lysosomes. The enzyme was characterized by RIA with an antibody specific for ET-1 and also by reverse-phase HPLC. For both sources, the pH rate profile of the membrane fraction had a very sharp maximum at pH 7.0; little or no activity was seen at more acidic pH values. In contrast, the cytosolic fraction had a major peak at acidic pH values, as well as a broad peak in the neutral region. The activity at pH 7.0 in the membrane fraction was shown by reverse-phase HPLC to produce ET-1 and C-terminal fragment as products. This activity was abolished by phosphoramidon, EDTA, and 1,10-phenanthroline but was not inhibited by pepstatin A, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, or E-64--consistent with the characteristics of a metalloprotease. These results suggest that this activity is from the physiologically relevant, phosphoramidon-inhibitable, endothelin-converting enzyme. The activity found at neutral pH values in the cytosolic fraction was only partially inhibited by EDTA and 1,10-phenanthroline but was not inhibited by phosphoramidon. The membrane-bound endothelin-converting enzyme from human umbilical vein endothelial cells and bovine aortic endothelial cells showed marked similarities, including IC50 values for phosphoramidon of 2.7 and 1.8 microM and Km values for big-ET-1 of 45.4 and 20.9 microM, respectively. The apparent molecular mass by gel filtration was approximately 300-350 kDa for the enzyme from either source. This report characterizes human endothelin-converting enzyme, which may be an important therapeutic target for cardiovascular disease.     

A microsomal endoprotease that specifically cleaves isoprenylated peptides.

A microsomal enzymatic activity is described that can specifically cleave the tetrapeptide N-acetyl-S-farnesyl-L-Cys-L-Val-L-Ile-L-Ser between the isoprenylated cysteine residue and the valine residue. Km and Vmax values are measured as 5.8 microM and 251 pmol/min per mg of protein, respectively. Proteolytic cleavage of the substrate is stereospecific because the substitution of a farnesylated D-cysteine residue for the L-amino acid leads to the abolition of substrate activity. A free carboxyl-terminal group is also required for substrate activity because methyl esterification renders the substrate inert. The tripeptide N-acetyl-S-farnesyl-L-Cys-L-Val-L-Ile and the dipeptide N-acetyl-S-farnesyl-L-Cys-L-Val are also hydrolyzed by the protease. Again, stereospecificity is observed at the isoprenylated residue. Hydrolysis of the farnesylated tetrapeptide is not inhibited by a 5-fold excess of the nonfarnesylated tetrapeptide, suggesting that isoprenylation is important for substrate activity. This activity is probably the same as the proteolytic activity proposed to cleave isoprenylated proteins terminating in a Cys-Ali-Ali-Xaa motif, where Ali refers to aliphatic amino acid. These proteins include the ras family of G proteins and the heterotrimeric G proteins. Proteolytic maturation of these essential isoprenylated signal-transducing elements is a key step in their activation.     

RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.

In the yeast Saccharomyces cerevisiae, mutations in either of two unlinked genes, RAM1 or RAM2, abolish the farnesyltransferase activity responsible for prenylation of Ras proteins and the a-factor mating pheromone. Here we report that the function of RAM1 and RAM2 genes is required for the membrane localization of Ras proteins and a-factor. The RAM2 gene was sequenced and can encode a 38-kDa protein. We examined the functional interaction of RAM2 and RAM1 by expressing the genes in Escherichia coli. Extracts derived from an E. coli strain that coexpressed RAM1 and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates. In contrast, extracts derived from E. coli strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferase activity was reconstituted. These results indicate that the yeast farnesyl-protein transferase is comprised of Ram1 and Ram2 polypeptides. Although Ram1 is a component of the enzyme, disruption of the RAM1 gene in yeast was not lethal, indicating that the Ram1-Ram2 farnesyltransferase is not essential for viability. In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Ram1 in protein farnesylation.     

A 170-kDa membrane-bound protease is associated with the expression of invasiveness by human malignant melanoma cells.

Malignant spreading of cancer cells requires cell surface proteases that cleave the crosslinked collagenous matrix of connective tissues. From correlating the morphologically defined invasiveness of tumor cells with the presence of specific membrane-associated proteases, we have identified a malignant human melanoma cell line, LOX, that invades crosslinked gelatin films in vitro and contains uniquely a neutral 170-kDa gelatinase in the cell membrane. A similar gelatinase was found in membranes recovered from culture media conditioned with LOX. The 170-kDa gelatinase is a wheat germ agglutinin-binding protein. The proteolytic activity is maximal at neutral pH, enhanced by EDTA and dithiothreitol, inhibited by the cysteine protease inhibitors N-ethylmaleimide, HgCl2, and phenylmethylsulfonyl fluoride, and can bind to an organomercurial adsorbent, suggesting that it is a neutral sulfhydryl-sensitive protease. This 170-kDa gelatinase of LOX cells was not found in a control melanoma cell line, SK-MEL28, or in 32 other tumor cell lines that did not show extracellular gelatin degradation. Thus, we have identified a large membrane-bound protease that may be a specific marker molecule for melanoma cell invasiveness.     

RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody

Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.     

Proteolytic activities in hypertensive cardiomyopathy of rats

Two-kidney, one clip Goldblatt hypertension of 2, 4 and 8 weeks duration was induced in 100-g male Wistar-Kyoto rats. Nucleic acid content was determined in the isolated cardiac muscle cells from the left ventricle. The profile for several major proteolytic activities in either isolated cardiac muscle cells or left ventricle preparations was also studied, using [3H]acetyl-casein as substrate. From the soluble fraction of the tissue or cell preparation, a pH 6 proteolytic activity, two forms of calcium-activated protease as well as cathepsin D were identifiable by inhibitor assay or DEAE-cellulose chromatography. From the myofibrillar fraction of the same preparation, two kinds of proteolytic activity were detected at alkaline pH: a phenylmethylsulfonyl fluoride (PMSF) inhibitable activity that was serine protease-like and the other a N-ethylmaleimide (NEM) inhibitable activity that resembled Ca2+-activated protease. At 2 weeks of hypertension, there was a significant increase in the pH 6 proteolytic activity as well as the calcium-activated protease I and the NEM-inhibitable alkaline protease activities, while the other identifiable proteolytic activities remained unchanged. Lysosomal cathepsin D showed a rise in activity only after 8 weeks of hypertension. These results may be related to the development of myocyte necrosis and lysis that occur in this model of hypertensive cardiomyopathy.     

Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo.     

p21ras is modified by a farnesyl isoprenoid.

Association of oncogenic ras proteins with cellular membranes appears to be a crucial step in transformation, ras is synthesized as a cytosolic precursor, which is processed to a mature form that localizes to the plasma membrane. This processing involves, in part, a conserved sequence, Cys-Ali-Ali-Xaa (in which Ali is an amino acid with an aliphatic side chain and Xaa is any amino acid), at the COOH terminus of ras proteins. Yeast a-factor mating hormone precursor also possesses a COOH-terminal Cys-Ali-Ali-Xaa sequence. However, while the COOH-terminal cysteine has been implicated as a site of palmitoylation of ras proteins, in mature a-type mating factor this residue is modified by an isoprenoid, a farnesyl moiety. We asked whether the Cys-Ali-Ali-Xaa sequence signaled different modifications for the yeast peptides (farnesylation) than for ras proteins (palmitoylation) or whether ras proteins were similar to the mating factors and contained a previously undiscovered isoprenoid. We report here that the processing of ras proteins involves addition of a farnesyl moiety, apparently at the COOH-terminal cysteine analogous to the cysteine modified in the yeast peptides, and that farnesylation may be important for membrane association and transforming activity of ras proteins.     

Ubiquitin has intrinsic proteolytic activity: implications for cellular regulation.

Ubiquitin is a protein of 76 amino acids found in every eukaryotic cell. Although ubiquitin is implicated in ATP-dependent nonlysosomal protein degradation and is also conjugated to specific cellular proteins, the role played by ubiquitin in cellular events has not been defined. We report that purified ubiquitin has intrinsic proteolytic activity and demonstrate that this activity is comparable to that of other well-characterized proteases. Monoclonal antibodies specific to ubiquitin inhibit proteolysis. Ubiquitin has protease activity over a broad pH range with an optimum at pH 8.0. It is stimulated by Ca2+ and is inhibited by high concentrations of phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. Ubiquitin will cleave proteins at a limited number of sites. We propose that the ubiquitination of a protein can convert that protein into an ad hoc specific protease and models are presented as to how this can play a role in regulating a variety of cellular events.     

Identification of calcium-activated neutral protease as a processing enzyme of human interleukin 1 alpha.

We describe here the involvement of calcium-activated neutral protease (CANP or calpain, EC 3.4.22.17) in calcium-dependent proteolytic processing of the precursor of human interleukin 1 alpha (IL-1 alpha) into mature IL-1 alpha. Calcium ionophore ionomycin enhanced proteolytic processing of pre-IL-1 alpha and the release of mature IL-1 alpha either from lipopolysaccharide (LPS)-activated human adherent mononuclear cells or from a human bladder carcinoma cell line (HTB9 5637) that constitutively produces human IL-1 alpha and -beta. The proteolytic processing of pre-IL-1 alpha was completely inhibited by EGTA. Similar calcium-dependent proteolytic processing of pre-IL-1 alpha was also observed with lysates of either LPS-activated human adherent mononuclear cells or HTB9 5637 cells. Since the optimal pH for processing was between 7 and 8, and E-64 (a cysteine protease inhibitor) and leupeptin (a serine and cysteine protease inhibitor) both inhibited this processing by cell lysates, we hypothesized that a calcium-activated neutral protease, CANP, might be responsible for this processing. This hypothesis was supported by data showing that the specific CANP inhibitor peptide inhibited this proteolysis in cell lysates in a dose-dependent fashion (IC50 = 0.05 microM) and that treatment of pre-IL-1 alpha with purified CANP yielded the 17-kDa mature form of IL-1 alpha, which has an amino terminus identical with that reported for mature human IL-1 alpha. Taken together, these findings indicate that calcium-dependent proteolytic processing of pre-IL-1 alpha is selectively mediated by CANP.     

Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.

The channel-forming activity of colicin E1 in artificial membranes is known to increase at low pH values and to have a maximum near pH 4 in such membrane vesicles. The present work demonstrates that this pH dependence of activity can be attributed to membrane binding. Maximal binding of colicin E1 and a more slowly binding channel-forming carboxyl-terminal tryptic peptide occurred at acidic pH values, with the effective pK values for binding equal to 4.6 and less than 4.0, respectively. The binding did not require imposition of a transmembrane potential. Insertion of the tryptic peptide into the membrane was shown by retention of bound [3H]leucine-labeled peptide by vesicles after digestion with protease, as well as by retention of the peptide in salt-washed vesicles. The retention after protease treatment was also used to estimate the amount of carboxyl-terminal peptide inserted into the membrane. Approximately 12 of the 21 leucines present in the carboxyl-terminal peptide were retained after Pronase treatment at pH less than 4. Reversibility of the insertion at low pH values was seen after an alkaline shift of pH to 6.0, resulting in a decrease of the protease-inaccessible fraction of the bound protein. A model is presented describing a mechanism in which protonation of one or more carboxyl residues is necessary for effective binding and insertion into the membrane by the channel-forming domain of colicin E1. This model may also be relevant to the mechanism of membrane insertion by certain toxins.     

The gamma subunit of transducin is farnesylated.

Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification.     

Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze compartment-specific membrane fusion. Whereas most SNAREs are bona fide type II membrane proteins, Ykt6 lacks a proteinaceous membrane anchor but contains a prenylation consensus motif (CAAX box) and exists in an inactive cytosolic and an active membrane-bound form. We demonstrate that both forms are farnesylated at the carboxyl-terminal cysteine of the CCAIM sequence. Farnesylation is the prerequisite for subsequent palmitoylation of the upstream cysteine, which permits stable membrane association of Ykt6. The double-lipid modification and membrane association is crucial for intra-Golgi transport in vitro and cell homeostasis/survival in vivo. The membrane recruitment and palmitoylation is controlled by the N-terminal domain of Ykt6, which interacts with the SNARE motif, keeping it in an inactive closed conformation. Together, these results suggest that conformational changes control the lipid modification and function of Ykt6. Considering the essential and central role of Ykt6 in the secretory pathway, this spatial and functional cycle might provide a mechanism to regulate the rate of intracellular membrane flow.     

Expression and characterization of the Babesia bigemina cysteine protease BbiCPL1

BbiCPL1 was the first papain-like cysteine protease from a piroplasm to be identified with proteolytic activity. Here we report the improved production of the active recombinant enzyme, and the biochemical characterization of this potential drug target. BbiCPL1 showed characteristic properties of its class, including hydrolysis of papain-family peptide substrates, an acidic pH optimum, requirement of a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors such as E-64, leupeptin, ALLN and cystatin. The optimum pH for the protease activity against peptide substrates was 5.5, but enzymatic activity was observed between pH 4.0 and pH 9.0. At slightly basic pH 7.5, BbiCPL1 maintained 83% of maximum activity, suggesting a role in cytosol environment.     

Proteolysis patterns of epitopically labeled yeast DNA topoisomerase II suggest an allosteric transition in the enzyme induced by ATP binding.

A cloned yeast TOP2 gene was modified to produce yeast DNA topoisomerase II (EC 5.99.1.3) epitopically labeled at its amino or carboxyl terminus. Limited digestion with SV8 endoprotease shows three distinct protease-sensitive sites in each polypeptide of the dimeric enzyme. These sites were mapped by immunostaining of the end-labeled proteolytic fragments resolved by SDS/polyacrylamide gel electrophoresis; two of the mapped locations were confirmed by sequencing the amino ends of two unlabeled peptic fragments. Proteolytic cleavage by SV8 endoprotease at a pair of sites corresponding to the carboxyl sides of Glu-411 and Glu-680 is modulated by the binding of the nonhydrolyzable ATP analogs adenosine 5'-[beta, gamma-imido]triphosphate (5'-adenylyl imidodiphosphate) and adenosine 5'-[gamma-thio]triphosphate: in their absence cleavage occurs predominantly at Glu-411; in the presence of either analog, cleavage occurs predominantly at Glu-680. These results are interpreted in terms of allosteric interdomainal movements in the type II DNA topoisomerase following the binding of ATP.