SEARCH · Search PubMed

Results for “Hydrolysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed2006

A "moonlighting" dizinc aminopeptidase from Streptomyces griseus: mechanisms for peptide hydrolysis and the 4 x 10(10)-fold acceleration of the alternative phosphodiester hydrolysis.

A unique "enzyme catalytic promiscuity" has recently been observed, wherein a phosphodiester and a phosphonate ester are hydrolyzed by a dinuclear aminopeptidase and its metal derivatives from Streptomyces griseus (SgAP) [Park, H. I., Ming, L.-J. (1999) Angew. Chem., Int. Ed. Engl. 38, 2914-2916 and Ercan, A., Park, H. I., Ming, L.-J. (2000) Chem. Commun. 2501-2502]. Because tetrahedral phosphocenters often serve as transition-state inhibitors toward the hydrolysis of the peptide, phosphoester hydrolysis by peptidases is thus not expected to occur effectively and must take place through a unique mechanism. Owing to the very different structures and mechanistic requirements between phosphoesters and peptides during hydrolysis, the study of this effective phosphodiester hydrolysis by SgAP may provide further insight into the action of this enzyme that is otherwise not obtainable from regular peptide substrates. We present herein a detailed investigation of both peptide and phosphodiester hydrolyses catalyzed by SgAP. The latter exhibits a first-order rate enhancement of 4 x 10(10)-fold compared to the uncatalyzed reaction at pH 7.0 and 25 degrees C. The results suggest that peptide and phosphodiester hydrolyses by SgAP may share a common reaction mechanism to a certain extent. However, their differences in pH dependence, phosphate and fluoride inhibition patterns, and proton inventory reflect that they must follow different pathways. Mechanisms for the two hydrolyses are drawn on the basis of the results, which provide the foundation for further investigation of the catalytic promiscuity of this enzyme by means of physical and molecular biology methods. The catalytic versatility of SgAP suggests that this enzyme may serve as a unique "natural model system" for further investigation of dinuclear hydrolysis. A better understanding of enzyme catalytic promiscuity is also expected to shed light on the evolution and action of enzymes.

Amino Acid Sequence↗

PubMedDate not supplied

Hydrolysis of poly(alkylene amidophosphate)s containing amino acid or peptide residues in the side groups. Kinetics and selectivity of hydrolysis.

Kinetics of hydrolysis of poly(alkylene amidophosphate)s with amino acids or dipeptides as the side groups was studied by 31P NMR at pH 1.5, 6.5, and 8.5. The direction of hydrolysis and the relative rate coefficients of breaking P-O bonds in the main chain and P-N bonds in the side groups depend strongly on the pH of the medium of hydrolysis. The P-N (amide) bond hydrolyzes much faster than the P-O (ester) bond in acidic and close to neutral conditions (negligible P-O hydrolysis), whereas above pH > or = 8.5 these differences are much smaller. For instance, for 4-Ala the rate coefficients of hydrolysis are equal (in H2O at 37 degrees C and pH 8.5) to 1.9 x 10(-8) s(-1) and 1.0 x 10(-9) s(-1) for the P-N and P-O bonds, respectively, quite different from the values found for the low molecular model 2 (0 and 1.4 x 10(-7) s(-1), respectively).

Amides↗

PubMed1993

Hydrolysis of partially saturated egg phosphatidylcholine in aqueous liposome dispersions and the effect of cholesterol incorporation on hydrolysis kinetics.

Hydrolysis kinetics of partially hydrogenated egg phosphatidylcholine (PHEPC) were studied as a function of pH, temperature, buffer concentration, ionic strength, and the effect of cholesterol incorporation. Results showed that PHEPC has a maximum stability at around pH 6.5. General acid base catalysis was observed for acetate, HEPES and Tris buffers. Increasing the ionic strength of the buffer solutions did not influence the hydrolysis kinetics. The relationship between the observed hydrolysis rate constants and the temperature could adequately be described by the Arrhenius equation. Incorporation of cholesterol did not affect the hydrolysis kinetics. This result indicates that the hydrolysis kinetics of PHEPC do not depend on the changes in bilayer rigidity induced by cholesterol incorporation. Cholesterol is stable under the experimental conditions used in this study; no changes were observed in cholesterol concentration over the experimental time interval.

Buffers↗

PubMed1990

[Characteristics of kinetics of phospholipid hydrolysis by phospholipase C from Bacillus cereus. Hydrolysis of phosphatidylcholine in the presence of deoxycholate].

Using dynamic light scattering and 31P-NMR spectroscopy methods, the reaction of solubilization of phosphatidylcholine by the ionic detergent, sodium deoxycholate, in aqueous solutions was studied. The kinetics of phosphatidylchodine hydrolysis by phospholipase C from B. cereus depending on the size and structural organization of substrate aggregates was investigated. No phosphatidylcholine hydrolysis was observed in the case of lamellar organization of the substrate, the size of lamellas not exceeding 2000-5000 A. The substrate hydrolysis rate within mixed micelles was controlled by the accessibility of the substrate on the surface of micellar aggregates. There was a decrease in the phosphatidylcholine hydrolysis rate at high detergent concentrations in the system. It was concluded that such a decrease in the hydrolysis rate can be due to two reasons, i) the decrease in mixed micelle size with a simultaneous decrease of surface concentration of the substrate, and, ii) the formation of "pure" detergent micelles capable to adsorb the enzyme by decreasing the "effective" concentration of phospholipase C.

Bacillus cereus↗

PubMedDate not supplied

Effect of P-450 inducers on biliary excretion of glutathione and its hydrolysis products. Correlation between hepatic gamma-glutamyltranspeptidase activity and the proportion of glutathione hydrolysis products in bile.

This study was designed to determine if a relationship exists between hepatic gamma-glutamyltranspeptidase (gamma-GT) activity and the biliary excretion of glutathione (GSH) and its hydrolysis products. Rats were pretreated with the following microsomal enzyme inducers: pregnenolone-16 alpha-carbonitrile (PCN), dexamethasone (DEX), 3-methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phenobarbital (PB), ethanol (ETOH), trans-stilbene oxide (TSO), butylated hydroxyanisole (BHA), isosafrole (ISF), clofibrate, and benzo(a)pyrene. Hepatic gamma-GT activity was quantitated spectrophotometrically; bile and liver samples were analyzed by HPLC for reduced and oxidized GSH and their hydrolysis products (cysteine, cysteinylglycine, and cysteinylglycine disulfide). Administration of the inducers had only minor effects on hepatic GSH concentration, as BHA was the only agent to increase GSH concentration. However, these inducers had a pronounced effect on the biliary excretion of total thiol-derived sulfur as PCN, PB, and ISF produced an increase, whereas TCDD, ETOH, and TSO caused a decrease. The relative amount of the GSH hydrolysis products in bile was highly dependent on gamma-GT activity. For example, hepatic gamma-GT activity was increased by PCN, DEX, BHA, TSO, and ISF. They also increased the GSH hydrolysis products to total thiol-derived sulfur ratio in bile. In conclusion, the ratio of GSH hydrolysis products to total thiol-derived sulfur excreted in rat bile reflects the hepatic gamma-GT activity.

Animals↗

PubMed1993

[Characteristics of phospholipid hydrolysis kinetics by phospholipase C from Bacillus cereus. Hydrolysis of phosphatidylinositol in various aggregated states].

The process of phospholipase C hydrolysis of phosphatidylethanolamine (PE) in the form of mixed micelles phospholipid-detergent or in the form of vesicules in the mixture with phosphatidylcholine (PC) was studied. The size of the micelles was measured by dynamic light-scattering and their structure was determined by 31P-NMR spectroscopy. It was found that the kinetics of PE hydrolysis in the micelles by phospholipase C from Bacillus cereus do not follow Michaelis-Menten equation, but at all concentrations studied PE was hydrolyzed significantly slower than PC. The rate of PC hydrolysis was measured in the previous work. The hydrolysis of PE in PC-vesicules was followed with use of Victoria blue R dye method. It was shown that the rate of PE hydrolysis in the vesicules is similar to that of PC.

Bacillus cereus↗

PubMed1999

Hydrolysis of a Model for the 5'-Cap of mRNA by Dinuclear Copper(II) and Zinc(II) Complexes. Rapid Hydrolysis by Four Copper(II) Ions.

Two bis(triazacyclononane) ligands, 1,3-bis(1,4,7-triaza-1-cyclononyl)-p-xylene (pXTD) and 1,3-bis(1,4,7-triaza-1-cyclononyl)-m-xylene (mXTD), form stable dinuclear Cu(II) complexes (Cu(2)L). At pH 7.3, the predominant species are bis(hydroxide) complexes (Cu(2)L(OH)(2)(2+)) as determined by equilibrium modeling of pH-potentiometric measurements. Several dinuclear and mononuclear Cu(II) complexes and a dinuclear Zn(II) complex promote the hydrolysis of GpppG, a model for the 5'-cap of mRNA. At 0.125 mM complex, both Cu(2)(pXTD) and Cu(2)(mXTD) promote hydrolysis of GpppG approximately 100-fold more rapidly than does the monomeric Cu(TACN) complex (0.250 mM) at pH 7.3 and 37 degrees C (TACN = 1,4,7-triazacyclononane). The dependence of the rate constant on dinuclear Cu(II) complex concentration suggests that Cu(2)(pXTD) promotes hydrolysis through both a 1:1 complex (Cu(2)L-GpppG) and a 2:1 complex ((Cu(2)L)(2)-GpppG). The 2:1 complex is 20-fold more reactive than the 1:1 complex; a first-order rate constant of 1.1 x 10(-)(4) s(-)(1) is determined for hydrolysis of the 2:1 complex. Cu(2)(mXTD) effectively promotes the hydrolysis of GpppG only through a 2:1 complex which hydrolyzes with a first-order rate constant of 4.3 x 10 (-)(5) s(-)(1). Cu(2)(pXTD) binds as a 1:1 complex to m(7)GpppG with a binding constant of 27 000 M(-)(1) as determined by use of fluorescence spectroscopy. Two Cu(2)(mXTD) complexes bind stepwise to m(7)GpppG with binding constants of 5300 and 12 000 M(-)(1) for the first complex and second complex, respectively.

Journal Article↗

PubMed1981

Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Characterization of ATP hydrolysis.

Both single- and double-stranded DNA stimulate the hydrolysis of ATP catalyzed by the recA protein of Escherichia coli. However, the reactions differ in their pH optima, response to recA protein concentration, salt sensitivity, and degree of inhibition by ADP, all of which reflect different requirements for the prehydrolytic binding of single- and double-stranded DNA by the RecA protein. Single- and double-stranded DNA stimulate hydrolysis of the same nucleoside triphosphates, principally (r,d)ATP and (r,d)UTP, suggesting that a single hydrolytic site is utilized in both single- and double-stranded DNA-dependent reactions. recA protein also catalyzes detectable ATP hydrolysis in the absence of exogenous DNA, although the rate is reduced 2 to 3 orders of magnitude. This DNA-independent hydrolysis shows the same nucleotide specificity at pH 6.2 and 7.5, although the rate of hydrolysis depends upon the pH.

Adenosine Triphosphate↗

PubMed2002

Kinetic peculiarities of human tissue kallikrein: 1--substrate activation in the catalyzed hydrolysis of H-D-valyl-L-leucyl-L-arginine 4-nitroanilide and H-D-valyl-L-leucyl-L-lysine 4-nitroanilide; 2--substrate inhibition in the catalyzed hydrolysis of N alpha-p-tosyl-L-arginine methyl ester.

Hydrolysis of D-valyl-L-leucyl-L-lysine 4-nitroanilide (1), D-valyl-L-leucyl-L-arginine 4-nitroanilide (2), and N alpha-p-tosyl-L-arginine methyl ester (3) by human tissue kallikrein was studied throughout a wide range of substrate concentrations. At low substrate concentrations, the hydrolysis followed Michaelis-Menten kinetics but, at higher substrate concentrations, a deviation from Michaelis-Menten behavior was observed. With the nitroanilides, a significant increase in hydrolysis rates was observed, while with the ester, a significant decrease in hydrolysis rates was observed. The results for substrates (1) and (3) can be accounted for by a model based on the hypothesis that a second substrate molecule binds to the ES complex to produce a more active or an inactive SES complex. The deviation observed for substrate (2) can be explained as a bimolecular reaction between the enzyme-substrate complex and a free substrate molecule.

Catalysis↗

PubMed1986

Hydrolysis of cyclic nucleotides by a purified cGMP-stimulated phosphodiesterase: structural requirements for hydrolysis.

Hydrolysis of cyclic AMP and cyclic GMP analogues by a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue was investigated by reversed-phase HPLC. The results indicate that both a negative charge and an equatorial oxygen atom located at the cyclic phosphate residue are absolute requirements for the process of hydrolysis. Other substituents only gradually decreased the apparent hydrolytic activity. C-8-substituted derivatives were generally poor substrates due to the limited ability of these compounds to rotate freely around the glycosidic bond. While C-6- and 0-2'-substituted analogues carrying bulky substituents were also poorly hydrolysed, all other derivatives, including different C-2-, C-6-, 0-3'- and 0-5'-modified cyclic nucleotides, were good substrates. We consistently observed that cyclic GMP and cyclic GMP analogues were better hydrolysed than the corresponding cyclic AMP analogues. Hydrolysis was correlated with neither the hydrogen bond donor/acceptor abilities nor the hydrophobicity of selected cyclic nucleotide analogues. Based on quantum-chemical calculations of the size and direction of the dipole moments of different purine bases, we propose that the polarization of inducible amino acid side-chains within the binding site is involved in the differential binding of adenine-derived and guanine-derived nucleotides. However, the size of the dipole moment alone is not sufficient to explain the observed cGMP-preference. Rather, the direction of the polarization power relative to the other molecular structures involved in binding and hydrolysis seems to be the molecular mechanism by which the enzyme is able to discriminate between cAMP- and cGMP-like structures.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1985

Phosphatidylinositol hydrolysis and phosphatidylinositol 4',5-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas.

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca2+-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca2+ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of 32PO4 into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca2+-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This 32PO4 turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4',5'-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca2+ deficiency. This phosphatidylinositol 4',5'-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca2+ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4',5'-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4',5'-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca2+ mobilization.

Animals↗

PubMed1999

The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins.

BACKGROUND: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.

Crystallography, X-Ray↗

PubMed1998

Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 2. Kinetic mechanism for the sequential hydrolysis of two ATP.

In the preceding paper, we showed that DNA topoisomerase II from Saccharomyces cerevisiae binds two ATP and rapidly hydrolyzes at least one of them before encountering a slow step in the reaction mechanism. These data are potentially consistent with two different types of reaction pathways: (1) sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP. Here, we present results that are consistent only with topoisomerase II hydrolyzing its two bound ATP sequentially. Additionally, these results indicate that the products of the first hydrolysis are released from the enzyme before the second ATP is hydrolyzed. Release of products from both the first and second hydrolyses contributes to the rate-determining process. The proposed mechanism for ATP hydrolysis by topoisomerase II is complex, having nine rate constants. To calculate values for each of these rate constants, a technique of kinetic parameter estimation was developed. This technique involved using singular perturbation theory in order to estimate rate constants, and consequently identify kinetic steps following the rate-determining step.

Adenosine Diphosphate↗

PubMed1984

Mechanistic studies on metal-pyrophosphate hydrolysis. Structure of tetraammine(chloroaquo)cobalt (III) dichloride ([Co(NH3)4ClH2O]2+.2Cl-), an acid hydrolysis product of Co(NH3)4HP2O7 and Co(NH3)4PO4.

The octahedral complex tetraammine(chloroaquo)cobalt(III) dichloride is shown to be the HCl hydrolysis product of both P1,2-bidentate tetraammine(pyrophosphato)cobalt(III) [Co(NH3)4HP2O7 or CoPP] and bidentate tetraammine(phosphato)cobalt(III) [Co(NH3)4PO4 or CoP]. The complex crystallizes in the orthorhombic space group Pna21 with cell dimensions a = 13.033(2)A, b = 6.710(1)A, and c = 10.318(2)A; the crystal structure was refined to a final disagreement index of 0.033. The average of the four Co-N distances is 1.944 +/- 6A. The Co-Cl distance is 2.257(2)A and the Co-O(W) distance is 1.971(4)A. Both protons of the coordinated water molecule are engaged in strong hydrogen bonds to the two nonbonded chloride counterions with O(W)-Cl distances of 3.087(6)A and 3.123(6)A. Each nonbonded chloride is engaged in seven hydrogen bonding interactions resulting from the high ratio of hydrogen bond donors to acceptors in the CoP structure. Cobalt bisphosphate (CoP2) is the final enzyme hydrolysis product when CoPP is used as substrate in the yeast inorganic pyrophosphatase reaction. The bridge oxygen atom is the site of initial CoPP cleavage both for HCl catalyzed hydrolysis as well as for enzyme catalyzed hydrolysis.

Cobalt↗

PubMed1994

The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice.

The standard free energy for hydrolysis of the GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP), which is -5.18 kcal in solution, was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtubules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that the majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described here were obtained from video microscopy measurements of the kinetics of assembly and disassembly of GMPCPP-microtubules and GMPCP-microtubules. It was possible to study GMPCPP-microtubules since GMPCPP is not hydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassium) Pipes buffer with glycerol, which reduced the half-time for GMPCPP hydrolysis from > 10 h to approximately 10 min. The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s-1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.

Animals↗

PubMed1995

CD8-dependent CTL require co-engagement of CD8 and the TCR for phosphatidylinositol hydrolysis, but CD8-independent CTL do not and can kill in the absence of phosphatidylinositol hydrolysis.

Most instances of MHC class I recognition and target cell killing by CD8+ CTL require the involvement of CD8. The role of CD8 in these events may be both for adhesion of the CTL with the APC, as well as for signal transduction through the TCR. The precise mechanism by which CD8 mediates signal transduction remains enigmatic. Similarly, it is unclear whether only the CD8 molecules which bind to the same class I molecule as the TCR contribute to signaling in the T cell responding to antigen. We have investigated the requirement for co-engagement of CD8 and the TCR in the induction of the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) during the interaction of CTL and APC transfected with either wild-type or mutant (CD8 non-binding) class I molecules. Our results show that for conventional CD8-dependent killing co-engagement of both CD8 and the TCR is required to initiate PIP2 hydrolysis. This requirement for co-engagement, however, can be overcome by a high density of ligand, such as that provided by high concentrations of exogenous peptide. In such situations, the binding of CD8 to non-antigenic class I molecules can elicit PIP2 hydrolysis. Therefore, during interactions between CTL and APC, which generally occur at low concentrations of antigenic peptide, triggering of PIP2 hydrolysis requires TCR and CD8 co-engagement, and the binding of CD8 to non-antigenic class I molecules does not contribute significantly to signaling within the T cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides.

Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it.

Adenosine Triphosphatases↗

PubMed1987

On the rate of F1-ATPase turnover during ATP hydrolysis by the single catalytic site. Evidence that hydrolysis with a slow rate of product release does not occur at the alternating active site.

Under conditions of molar excess of enzyme, isolated F1-ATPase from beef heart mitochondria catalyses ATP hydrolysis biphasically. The rate constants for product release are approximately 10(-1) and 10(-4)-10(-3) s-1, respectively. The slow phase of ATP hydrolysis is insensitive to EDTA. [gamma-32P]ATP splitting in the slow phase cannot be chased from the enzyme during several catalytic turnovers. It follows from these results that the slow single-site hydrolysis of ATP (kcat approximately 10(-4) s-1), initially observed by Grubmeyer et al. [(1982) J. Biol. Chem. 257, 12092-12100], is not carried out by the normal catalytic site. In contrast, the phase of rapid ATP hydrolysis (kcat approximately 10(-1) s-1) is completely prevented by EDTA and is believed to be the normal function of the normal catalytic site of F1-ATPase.

Adenosine Triphosphate↗