Effect of cadmium acetate on the conformation of lysozyme: functional implications

Structural variations of lysozyme as a consequence of its interaction with CdAc2, as well as the implications on the protein functionality have been studied. Variations in the conformation of the macromolecule are seen, however these changes are not reflected on the secondary structure. The interaction of the salt with the polypeptide chain is weak and thermodynamically unfavourable. Molecular aggregates (dimer forms) are observed at the highest salt concentrations. This interaction causes an inhibitory effect on lysozyme, the activity loss being 50% at the highest salt concentration studied. The inhibition is of mixed type with an uncompetitive component. Thus cadmium does not bind to the active site of the enzyme which is in accordance with the not very large activity loss observed. The substrate inhibition of lysozyme is favoured in the presence of the salt, so interaction with the macromolecule is at low affinity sites.     

The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.     

Effects of nucleotides on activity of a purified ADP-ribosyltransferase from turkey erythrocytes

A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADP-ribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mm nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mm ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, cAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20–200 μm) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.     

Lysosomal segregation of a mannose-rich glycoprotein imparted by the prosequence of myeloperoxidase

The role of the N-terminal sequence of myeloperoxidase in the intracellular targeting was examined by using glycosylated lysozyme as a reporter. A fusion protein was constructed in which the presequence residues −18 through −6 of the lysozyme moiety had been replaced by residues 1–158 of prepromyeloperoxidase. Expression of the fusion protein in Chinese hamster ovary cells demonstrated its partial secretion and partial intracellular retention. The latter was accompanied by trimming the myeloperoxidase prosequence off the lysozyme moiety. The rate of the retention of the lysozyme fusion protein was higher than that of glycosylated lysozyme that had been expressed in cells transfected with cDNA of glycosylated lysozyme. The retention was insensitive to NH₄Cl. In the secreted protein, lysozyme contained predominantly complex oligosaccharides as demonstrated by a proteolytic fragmentation in vitro and resistance to endo-β-N-acetylglucosaminidase H. In contrast, when targeted to lysosomes, the lysozyme moiety of the fusion protein contained predominantly mannose-rich oligosaccharides. In baby hamster kidney cells, the trimming of the oligosaccharides in the lysozyme fragment was less vigorous, and a selective targeting of molecules bearing mannose-rich oligosaccharides to lysosomes was more apparent than in Chinese hamster ovary cells. In the presence of monensin, the formation of complex oligosaccharides in the fusion protein and its secretion were strongly inhibited, whereas the intracellular fragmentation was not. We suggest that the prosequence of myeloperoxidase participates in the intracellular routing of the precursor and that this routing operates on precursors bearing mannose-rich rather than terminally glycosylated oligosaccharides and diverts them from the secretory pathway at a site proximal to the monensin-sensitive compartment of the Golgi apparatus. J. Cell. Biochem. 71:158–168, 1998. © 1998 Wiley-Liss, Inc.     

Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease.

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage lambda O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of lambda O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of lambda O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with lambda O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.     

Kinetics of protein substrate degradation by HslUV

The HslUV protease-chaperone complex degrades specific protein substrates in an ATP-dependent reaction. Current models propose that the HslU chaperone, a AAA protein of the Clp/Hsp100 family, binds and unfolds substrates and translocates the polypeptide into the catalytic cavity of the HslV protease. These processes are being characterized using substrates that are targeted to HslUV with a carboxy-terminal fusion of the natural substrate SulA or the carboxy-terminal 11 amino acid residues thereof. In a tandem fusion of green fluorescent protein with SulA, HslUV degrades the SulA moiety but not green fluorescent protein. Wild type and mutant Arc repressor variants are degraded; over a range of substrate stabilities, the specific rate of degradation and its dependence on substrate stability is similar to that of ClpXP. For a hyperstable Arc variant having an intermolecular disulfide bond, the rate of degradation by HslUV is an order of magnitude slower than by ClpXP. Similarity in degradation rates for a subset of substrates by HslUV and ClpXP suggests a similarity in mechanism of the apparent rate-limiting steps of unfolding and translocation by the chaperone components HslU and ClpX. The fall-off in degradation by HslUV for the more stable substrates that are degraded by ClpXP is consistent with the two systems acting on different spectra of biological substrates.     

Cloning and characterization of an Arabidopsis thaliana Nudix hydrolase homologous to the mammalian GFG protein

In a search for a plant antimutator MutT protein, an Arabidopsis thaliana Nudix hydrolase with homology to the mammalian GFG protein was expressed as a hexahistidine fusion polypeptide in Escherichia coli and purified to homogeneity. Unlike the GFG protein, the A. thaliana homolog could not complement the mutT mutation in a MutT-deficient E. coli strain nor was it able to hydrolyze 8-oxo-dGTP, the main substrate of the MutT protein. Instead the recombinant protein hydrolyzed a variety of nucleoside diphosphate derivatives showing a preference for ADP-ribose, with Km and k(cat) values of 1.2 mM and 2.7 s(-1) respectively. The products of ADP-ribose hydrolysis were AMP and ribose-5-phosphate. The optimal activity was at alkaline pH (8.5) with Mg2+ (5 mM) ions as the cofactor. The protein exists as a dimmer in solution.     

Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain).

A protein that greatly stimulates the multiple peptidase activities of the 20 S proteasome (also known as macropain, the multicatalytic protease complex, and 20 S protease) has been purified from bovine red blood cells and from bovine heart. The activator protein was a single polypeptide with an apparent molecular weight of 28,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a native molecular weight of approximately 180,000. This protein, which we have termed PA28, regulated all three of the putatively distinct peptidase activities displayed by each of two functionally different forms of the proteasome. This regulation usually included both an increase in the maximal reaction velocity and a decrease in the concentration of substrate required for half-maximal velocity and indicated that PA28 acted as a positive allosteric effector of the proteasome. PA28 failed, however, to stimulate the hydrolysis of large protein substrates such as casein and lysozyme. These results suggested that the hydrolysis of protein substrates occurred at a site or sites distinct from those that hydrolyzed small peptides and that the regulation of the two processes could be uncoupled. Evidence for direct binding of PA28 to the proteasome was obtained by glycerol density gradient centrifugation. PA28 may play an important regulatory role in intracellular proteolytic pathways mediated by the proteasome.     

Isosteric conversion of protein carboxyl groups into carboxamide groups. II. Application to lysozyme

For isosteric conversion of carboxyl groups of proteins into amide groups, ammonolysis of protein esters under mild conditions was attempted. Ammonolysis of methyl esters of lysozyme and bovine serum albumin proved to be incomplete. Highly reactive N-ethylsalicylamide esters of guanylated lysozyme were therefore prepared by subjecting the protein to reaction with N-ethylbenzisoxazolium ion at pH 4.2, 0 degree. Per molecule, 5-7 ester groups were introduced, with concomitant decrease of activity of 80-90%. Only 0.3 tyrosine was modified. On hydrolysis at pH 9.2 the activity was completely restored. At pH 7.9 three classes of ester groups could be distinguished: one group of high rate of hydrolysis (k1 = 1.5 min-1), three groups of intermediate rate (k2 = 0.13 min-1) and two groups of low rate (k3 = 0.018 min-1). The intermediate rate approximated the rate of hydrolysis of the model compound benzoylglycine N-ethylsalicylamide ester (k = 0.15 min-1). Ammonolysis at pH 9.2 in 2.0 M ammonia/ammonium acetate provided complete conversion of the ester groups into amide groups without restoration of activity, confirming the essentiality of certain carboxyl groups. In particular, rearrangement of the ester groups into relatively stable imide groups by O-N acyl migration was found to be completely absent. When native lysozyme was esterified with N-ethylbenzisoxazolium ion the activity did not completely return on hydrolysis.     

The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70

The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.     

Regulation of the functional activity of lysozyme by alkylhydroxybenzenes

In our study, we investigated the capacity of alkylhydroxybenzenes (AHB), which are microbial anabiosis autoinducers, for alteration of the enzymatic activity of the hen egg-white lysozyme, as well as the efficiency of hydrolysis of specific (peptidoglycan) and nonspecific (chitin) substrates catalyzed by lysozyme. AHB homologues (C7-AHB and C12-AHB), which differ in their hydrophobicity and effects in their interaction with lysozyme, were used as modifying agents. C7-AHB stimulated enzymatic activity within the whole range of concentrations used (10^?7?10^?3 M). More hydrophobic C12-AHB exhibited this ability only at low concentrations and inhibited fermentative activity at high concentrations, acting as a mixed-type inhibitor. Both AHB homologues caused changes in the hydrophobicity of lysozyme molecules. An increase in the affinity level between the C7-AHB-modified enzyme and the nonspecific substrate (colloidal chitin or cell wall polymers of Saccharomyces sp.) was observed, which manifested itself in the enhancement of the hydrolysis rate by 200–500% (as compared to the native enzyme). A significant effect on the efficiency of the lysozyme-catalyzed modifications of the substrate (peptidoglycan, colloidal chitin) structure as a result of its complexation with AHB was demonstrated. A stabilizing effect of C7-AHB and C12-AHB was revealed, which ensured a high level of activity of the AHB-modified enzyme (as compared to the control) after heat treatment (functional stability), as well as at nonoptimal temperatures of catalysis (operational stability). The biological significance of lysozyme modification with AHB and the practical aspects of its application are discussed.     

Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6

Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5'-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma.     

Purification and Partial Characterization of an Aminopeptidase from Lactobacillus lactis

A surface-bound aminopeptidase of Lactobacillus lactis cells was solubilized with lysozyme, and the extract was subjected to streptomycin sulfate precipitation, ammonium sulfate fractionation, chromatography on Sephadex G-100 and diethylaminoethyl-Sephadex A-50, and preparative polyacrylamide gel electrophoresis. The purified enzyme was homogeneous in disc electrophoretic analysis and consisted of a single polypeptide chain with a molecular weight of 78,000 to 81,000. The optimal pH and optimal temperature for enzyme activity were 6.2 to 7.2 and 47.5 degrees C, respectively, for l-lysine-4-nitroanilide as the substrate. The enzyme was activated by Co and Zn ions and inhibited by Cu, Hg, and Fe ions and by the metal-complexing reagents ethylenediaminetetraacetic acid, 1,10-phenanthroline, and alpha,alpha'-dipyridyl. Higher concentrations of substrate and hydrolysis products also inhibited the activity of the enzyme. The aminopeptidase had broad substrate specificity and hydrolyzed many amino acid arylamides and many peptides with unsubstituted NH(2)-terminal amino acids.     

Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6

Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5′-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma.     

p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.     

Mechanism of the targeting action of DnaJ in the DnaK molecular chaperone system

In the DnaK (Hsp70) molecular chaperone system of Escherichia coli, the substrate polypeptide is fed into the chaperone cycle by association with the fast-binding, ATP-liganded form of the DnaK. The substrate binding properties of DnaK are controlled by its two cochaperones DnaJ (Hsp40) and GrpE. DnaJ stimulates the hydrolysis of DnaK-bound ATP, and GrpE accelerates ADP/ATP exchange. DnaJ has been described as targeting the substrate to DnaK, a concept that has remained rather obscure. Based on binding experiments with peptides and polypeptides we propose here a novel mechanism for the targeting action of DnaJ: ATP.DnaK and DnaJ with its substrate-binding domain bind to different segments of one and the same polypeptide chain forming (ATP.DnaK)m.substrate.DnaJn complexes; in these ternary complexes efficient cis-interaction of the J-domain of DnaJ with DnaK is favored by their propinquity and triggers the hydrolysis of DnaK-bound ATP, converting DnaK to its ADP-liganded high affinity state and thus locking it onto the substrate polypeptide.     

Minimum substrate sequence for signal peptidase I of Escherichia coli

The minimum substrate sequence recognized by signal peptidase I (SPase I or leader peptidase) was defined by measuring the kinetic parameters for a set of chemically synthesized peptides corresponding to the cleavage site of the precursor maltose binding protein (pro-MBP). The minimum sequence of a substrate hydrolyzed by SPase I at a measurable rate was the pentapeptide Ala-Leu-Ala decreases Lys-Ile. The rates of hydrolysis of this substrate, however, were several hundred-fold lower than those observed for the maturation of MBP in Escherichia coli, suggesting that in addition to these minimal sites involved in recognition, other features of pro-MBP are also needed for the optimal rate of signal peptide cleavage by SPase I. One parameter may be the length of the polypeptide chain. Studies of the synthetic peptides showed that decreasing the length of the polypeptide chain of substrates decreased the substrate efficiency measured as kcat/Km. However, in one case a decrease in the length of a peptide corresponding to -7 to +3 positions of pro-MBP to a nonapeptide (-7 to +2) increased the substrate efficiency by about 900-fold. The nonapeptide is the most efficient substrate for the enzyme in vitro so far reported. It is speculated that better peptide substrates are the ones which are able to adopt folded structures.     

Association of a myristoylated protein with a biological membrane and its increased phosphorylation by protein kinase C

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM-cellulose cation-exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane-associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.     

Hydrolysis of soluble, linear, un-cross-linked peptidoglycans by endogenous bacterial N-acetylmuramoylhydrolases

Soluble, linear, uncross-linked peptidoglycans, prepared from two autolysis-defective mutants of Streptococcus faecium ATCC 9790 and from Micrococcus leuteus, were used as substrates for studies of hydrolysis by an N-acetylmuramoylhydrolase (muramidase). The kinetics of hydrolysis of these substrates and the ability of the muramidases isolated from S. faecium ATCC 9790 and from two autolysis-defective mutants, Lyt-14 and Aut-3, to carry out transglycosylation reactions were compared with the action of hen egg white lysozyme (EC 3.2.1.17). Hydrolysis of these substrates by the endogenous streptococcal muramidases resulted in the production of disaccharide-peptide monomers with the structure (formula; see text) as nearly the sole product. As estimated from increases in reducing groups, hydrolysis proceeded at a linear rate for extended intervals, with consumption of up to 75% of the substrate, even at substrate concentrations well below the Km value. Apparent Km and relative Vmax values for the three streptococcal enzymes were indistinguishable from each other or from those for hen egg white lysozyme. These results indicate that the autolysis-defective phenotype of these mutants cannot be attributed to differences in their muramidases. In contrast to the action of hen egg white lysozyme, the streptococcal muramidase failed to catalyze transglycosylations. The extended periods of hydrolysis at constant rates are consistent with the occurrence of multiple catalytic events after the formation of the enzyme-substrate complex.     

Processing of proteins by the molecular chaperone Hsp104

The molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotide binding or hydrolysis in the two nucleotide-binding domains (NBD) of Hsp104 and analyzed the consequences for chaperone function by monitoring ATP hydrolysis, polypeptide binding, polypeptide processing, and disaggregation. Our results reveal that ATP binding to NBD1 serves as a central regulatory switch for the chaperone; it triggers binding of polypeptides, and stimulates ATP hydrolysis in the C-terminal NBD2 by more than two orders of magnitude, implying that ATP hydrolysis in this domain is important for disaggregation. Moreover, we show that Hsp104 actively unfolds its polypeptide substrates during processing, demonstrating that AAA+ proteins involved in disaggregation share a common threading mechanism with AAA+ proteins mediating protein unfolding/degradation.