Permeable membrane/mass spectrometric measurement of solvent 1H/2H, 12C/13C, and 16O/18O kinetic isotope effects associated with alpha-chymotrypsin deacylation: evidence for reaction mechanism plasticity

We have measured, by permeable membrane/mass spectrometry, the 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects (kie) associated with acyl-alpha-chymotrypsin hydrolysis and transesterification. The hydrolysis of alpha-chymotrypsinyl 2-furoate has a 12C/13C kie of approximately 1.06. Transesterification of the same acyl enzyme shows 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects of 1.015 (0.003), 1.01-1.02, and 2.226 (0.007), respectively. From the temperature independence of the 16O/18O transesterification kinetic isotope effect and kinetic data reported elsewhere [Wang, C.-L. A., Calvo, K. C., & Klapper, M. H. (1981) Biochemistry 20, 1401-1408], we conclude that there are two active forms of acylchymotrypsin. We also propose that formation of the tetrahedral intermediate is the rate-limiting step in both hydrolysis and transesterification and that the position of the transition state in the transesterification is closer to the starting enzyme ester while that for the hydrolytic reaction is closer to the tetrahedral intermediate. These results are discussed in terms of reaction mechanism plasticity.     

The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction

The restriction endonuclease EcoRI hydrolyzes the Rp diastereomer of d(pGGsAATTCC), an analogue of d(pGGAATTCC) containing a chiral phosphorothioate group at the cleavage site between the deoxyguanosine and the deoxyadenosine residues (Connolly, B.A., Potter, B.V.L., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984) Biochemistry 23, 3343-3453). Performing the reaction in H2(18)O leads to d(pGG) and the hexanucleotide d([18O, S]pAATTCC) which has an 18O-containing phosphorothioate group at the 5' terminus. Further hydrolysis of this hexamer with nuclease P1 yields deoxyadenosine 5'-O-[18O]phosphorothioate which can be stereospecifically phosphorylated with adenylate kinase and pyruvate kinase to give Sp-[18O] deoxyadenosine 5'-O-(1-thiotriphosphate). 31P NMR spectroscopy shows the oxygen-18 in this compound to be in a bridging position between the alpha- and beta-phosphorus atoms. Thus, the hydrolysis reaction catalyzed by EcoRI proceeds with inversion of configuration at phosphorus. This result is compatible with a direct enzyme-catalyzed nucleophilic attack of H2O at phosphorus without involvement of a covalent enzyme intermediate.     

Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease.

The stereochemical course of the reaction catalyzed by the EcoRV restriction endonuclease has been determined. This endonuclease recognizes GATATC sequence and cuts between the central T and dA bases. The Rp isomer of d(GACGATsATCGTC) (this dodecamer contains a phosphorothioate rather than the usual phosphate group between the central T and dA residues, indicated by the s) was a substrate for the endonuclease. Performing this reaction in H2 18O gave [18O]dps(ATCGTC) (a pentamer containing an 18O-labeled 5'-phosphorothioate) which was converted to [18O]dAMPS with nuclease P1. This deoxynucleoside 5'-[18O]phosphorothioate was stereospecifically converted to [18O]dATP alpha S with adenylate kinase and pyruvate kinase [Brody, R. S., & Frey, P. A. (1981) Biochemistry 20, 1245-1251]. Analysis of the position of the 18O in this product by 31P NMR spectroscopy showed that it was in a bridging position between the alpha- and beta-phosphorus atoms. This indicates that the EcoRV hydrolysis proceeds with inversion of configuration at phosphorus. The simplest interpretation is that the mechanism of this endonuclease involves a direct in-line attack at phosphorus by H2O with a trigonal bipyramidal transition state. A covalent enzyme oligodeoxynucleotide species can be discounted as an intermediate. An identical result has been previously observed with the EcoR1 endonuclease [Connolly, B. A., Eckstein, F., & Pingoud, A. (1984) J. Biol. Chem. 259, 10760-10763]. X-ray crystallography has shown that both of these endonucleases contain a conserved array of amino acids at their active sites. Possible mechanistic roles for these conserved amino acids in the light of the stereochemical findings are discussed.     

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.     

Stereochemistry of the hydrolysis of adenosine 5'-thiophosphate catalyzed by venom 5'-nucleotidase

The stereochemical problem involving a pro-pro-prochiral phosphorus center, the hydrolysis of adenosine 5'-monophosphate to adenosine and inorganic phosphate catalyzed by the venom 5'-nucleotidase, has been studied by use of chiral [16O, 17O, 18O]thiophosphates (Psi). (Rp)- and (Sp)-[alpha-18O1]Adenosine 5'-thiophosphates (AMPS) were synthesized by a combined chemical and biochemical procedure. Hydrolysis of (Rp)- and (Sp)-[alpha-18O1]AMPS in H217O by 5'-nucleotidase gave two enantiomers of chiral Psi of unknown configuration. A 31P NMR method based on the combination of the quadrupolar effect of 17O [Tsai, M.-D. (1979) Biochemistry 18, 1468-1472] and the 18O isotope shift [Cohn, M., & Hu. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 200-203] has been developed to analyze the configuration of chiral Pso. The results indicate that hydrolysis of (Rp)- and (Sp)-[alpha-18O1]AMPS in H217O gave (R)- and (S)- [16O, 17O, 18O]Psi, respectively. Therefore the hydrolysis of AMPS catalyze by the venom 5'-nucleotidase must proceed with inversion of configuration at phosphorus, which suggests that the reaction is most likely an "in line" single displacement without involving a phosphoryl-enzyme intermediate and without pseudorotation.     

The transmembrane domain of subunit b of the Escherichia coli F1F(O) ATP synthase is sufficient for H(+)-translocating activity together with subunits a and c.

Subunit b is indispensable for the formation of a functional H(+)-translocating F(O) complex both in vivo and in vitro. Whereas the very C-terminus of subunit b interacts with F(1) and plays a crucial role in enzyme assembly, the C-terminal region is also considered to be necessary for proper reconstitution of F(O) into liposomes. Here, we show that a synthetic peptide, residues 1-34 of subunit b (b(1-34)) [Dmitriev, O., Jones, P.C., Jiang, W. & Fillingame, R.H. (1999) J. Biol. Chem.274, 15598-15604], corresponding to the membrane domain of subunit b was sufficient in forming an active F(O) complex when coreconstituted with purified ac subcomplex. H(+) translocation was shown to be sensitive to the specific inhibitor N,N'-dicyclohexylcarbodiimide, and the resulting F(O) complexes were deficient in binding of isolated F(1). This demonstrates that only the membrane part of subunit b is sufficient, as well as necessary, for H(+) translocation across the membrane, whereas the binding of F(1) to F(O) is mainly triggered by C-terminal residues beyond Glu34 in subunit b. Comparison of the data with former reconstitution experiments additionally indicated that parts of the hydrophilic portion of the subunit b dimer are not involved in the process of ion translocation itself, but might organize subunits a and c in F(O) assembly. Furthermore, the data obtained functionally support the monomeric NMR structure of the synthetic b(1-34).     

18O isotope exchange experiments on phospholipase A2 determined by 13C-NMR: monomeric phosphatidylcholine and micellar phosphatidylethanolamine substrates

Experiments were carried out to determine whether the hydrolytic step or the product release step is the rate-limiting step for non-activated phospholipase A2 hydrolysis (Dennis, E.A. (1983) in The Enzymes, 3rd Edn., Vol 16 (Boyer, P., ed.), pp. 307-353, Academic Press, New York) of mixed micelles of phosphatidylethanolamine and Triton X-100 in the absence of activator phospholipids and of monomeric short chain phosphatidylcholine in the absence of an interface (Lombardo, D. et al. (1986) J. Biol. Chem. 261, 11663-11666). Phospholipase A2-catalyzed exchange of H2(18)O into 1-alkyl-2-[1(13)C]lauroyl-sn-glycero-3-phosphorylethanolamine and into 1-hexanoyl-2-[1-13C]hexanoyl-sn-glycero-3-phosphorylcholine were examined. Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C-NMR. Both the substrate and products of the reactions were examined for 18O incorporation. 18O was incorporated into the fatty acid product, but no incorporation of 18O into the substrate was found. These results suggest that the hydrolytic step is not followed by a higher energy transition state and that it, or a step before it, is rate-limiting. Coupled with kinetic experiments, this strongly suggests that the hydrolytic step is the rate-limiting step. Thus, the role of micellar and membrane interfaces in phospholipase A2 reactions does not appear to be by aiding product removal from the enzyme active site.     

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.     

Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of the adenylosuccinate synthetase reaction as studied by positional isotope exchange

In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of various ligands. Positional isotope exchange occurred, as measured by nuclear magnetic resonance spectroscopy, when IMP was present. In the absence of IMP, with or without aspartate or succinate, the [gamma-18O]GTP did not exhibit scrambling. These results suggest that the adenylosuccinate synthetase reaction involves the participation of 6-phosphoryl-IMP as an obligatory intermediate. On the basis of experiments carried out in our laboratory as well as in others, we believe the GDP remains bound to the enzyme until the product, adenylosuccinate, is formed. All products may then dissociate randomly from the enzyme. The positional isotope exchange experiments, along with initial-rate experiments carried out in our laboratory, serve to explain the lack of partial exchange reactions associated with the synthetase (Fromm, H. J. (1958) Biochim. Biophys. Acta 29, 255-262), as well as the net inversion of configuration when chiral thio-GTP is converted to thiophosphate (Webb, M. R., Reed, G. H., Cooper, B. F., and Rudolph, F. B. (1984) J. Biol. Chem. 259, 3044-3046).     

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate.

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis

The epsilon-amino group of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase was postulated as the essential base which initiates catalysis by abstracting the proton at C-3 of ribulose 1,5-bisphosphate (Hartman, F. C., Soper, T. S., Niyogi, S. K., Mural, R. J., Foote, R. S., Mitra, S., Lee, E. H., Machanoff, R., and Larimer, F. W. (1987) J. Biol. Chem. 262, 3496-3501). To scrutinize this possibility, the site-directed Gly-166 mutant, totally devoid of ribulosebisphosphate carboxylase activity, was examined for its ability to catalyze each of three partial reactions. When carbamylated at Lys-191 (i.e. activated with CO2 and Mg2+), wild-type enzyme catalyzed the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon reaction intermediate of the carboxylase reaction (Pierce, J., Andrews, T. J., and Lorimer, G. H. (1986a) J. Biol. Chem. 261, 10248-10256). Likewise, when carbamylated at Lys-191, the Gly-166 mutant also catalyzed the hydrolysis of this reaction intermediate. The carbamylated wild type catalyzed the enolization of ribulose 1,5-bisphosphate as indicated by the transfer of 3H radioactivity from [3-3H]ribulose, 1,5-bisphosphate to the medium. However, even when carbamylated at Lys-191, the mutant protein did not catalyze the enolization of ribulose 1,5-bisphosphate. Additionally, unlike the decarbamylated wild-type enzyme, which catalyzed the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate in the absence of Mg2+, the mutant protein was inactive in this partial reaction. These properties exclude the epsilon-amino group of Lys-166 as an obligatory participant in the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. In contrast, these properties are consistent with the epsilon-amino group of Lys-166 functioning as an acid-base catalyst in the enolization of ribulose 1,5-bisphosphate (when the enzyme is carbamylated) and in the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (when the enzyme is decarbamylated). Alternatively, Lys-166 may stabilize the transition states of these two partial reactions.     

Role of the PR intermediate in the reaction of cytochrome c oxidase with O2

The first discernible intermediate when fully reduced cytochrome c oxidase reacts with O2 is a dioxygen adduct (compound A) of the binuclear heme iron-copper center. The subsequent decay of compound A is associated with transfer of an electron from the low-spin heme a to this center. This reaction eventually produces the ferryl state (F) of this center, but whether an intermediate state may be observed between A and F has been the subject of some controversy. Here we show, using both optical and EPR spectroscopy, that such an intermediate (P(R)) indeed exists and that it exhibits spectroscopic properties quite distinct from F. The optical spectrum of P(R) is similar or identical to the spectrum of the P(M) intermediate that is formed after compound A when two-electron-reduced enzyme reacts with O2. An unusual EPR spectrum with features of a CuB(II) ion that interacts magnetically with a nearby paramagnet [cf. Hansson, O., Karlsson, B., Aasa, R., V?nng?rd, T., and Malmstr?m, B.G (1982) EMBO J. 1, 1295-1297; Blair, D. F., Witt, S. N., and Chan, S. I. (1985) J. Am. Chem. Soc. 107, 7389-7399] can be uniquely assigned to the P(R) intermediate, not being found in either the P(M) or F intermediate. The binuclear center in the P(R) state may be assigned as having an Fe(a3)(IV)=O CuB(II) structure, as in both the P(M) and F states. The spectroscopic differences between these three intermediates are evaluated. The P(R) state has a key role as an initiator of proton translocation by the enzyme, and the thermodynamic and electrostatic bases for this are discussed.     

Enzyme-catalyzed DNA unwinding. The role of ATP in helicase III activity

The enzyme helicase III catalyzes ATP-dependent unwinding of double-stranded DNA (Yarranto, G. T., Das, R. H., and Gefter, M. L. (1979) J. Biol. Chem. 254, 11997-12001). The free enzyme is able to bind to double- and single-stranded DNA. In the presence of ATP the enzyme can bind single- but not double-stranded DNA. The enzyme catalyzes an ADP-ATP exchange reaction in the absence of DNA. It is suggested that there is an enzyme.phosphate complex that discriminates between the two forms of DNA. These results are discussed in relation to a model that accounts for catalytic unwinding of DNA coupled to ATP hydrolysis.     

Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (RP)- and (SP)-[18O]DPPE, respectively, as indicated by 31P nuclear magnetic resonance (NMR) analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configuration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[ 16O , 17O, 18O]phosphate ( [ 16O , 17O, 18O] DPPA ) with unknown configuration. The latter compound was then converted to 1-[ 16O , 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The configuration of the phosphopropane -1,2-diol was determined as RP by 31P NMR analysis following ring closure and methylation [ Buchwald , S. L., & Knowles, J. R. (1980) J. Am. Chem. Soc. 102, 6601-6603]. The results indicated that hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. Our results therefore support a two-step mechanism involving a phosphatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.     

Determination of the partial reactions of rotational catalysis in F1-ATPase

Steady-state ATP hydrolysis in the F1-ATPase of the F(O)F1 ATP synthase complex involves rotation of the central gamma subunit relative to the catalytic sites in the alpha3beta3 pseudo-hexamer. To understand the relationship between the catalytic mechanism and gamma subunit rotation, the pre-steady-state kinetics of Mg x ATP hydrolysis in the soluble F1-ATPase upon rapid filling of all three catalytic sites was determined. The experimentally accessible partial reactions leading up to the rate-limiting step and continuing through to the steady-state mode were obtained for the first time. The burst kinetics and steady-state hydrolysis for a range of Mg x ATP concentrations provide adequate constraints for a unique minimal kinetic model that can fit all the data and satisfy extensive sensitivity tests. Significantly, the fits show that the ratio of the rates of ATP hydrolysis and synthesis is close to unity even in the steady-state mode of hydrolysis. Furthermore, the rate of Pi binding in the absence of the membranous F(O) sector is insignificant; thus, productive Pi binding does not occur without the influence of a proton motive force. In addition to the minimal steps of ATP binding, reversible ATP hydrolysis/synthesis, and the release of product Pi and ADP, one additional rate-limiting step is required to fit the burst kinetics. On the basis of the testing of all possible minimal kinetic models, this step must follow hydrolysis and precede Pi release in order to explain burst kinetics. Consistent with the single molecule analysis of Yasuda et al. (Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., and Itoh, H. (2001) Nature 410, 898-904), we propose that the rate-limiting step involves a partial rotation of the gamma subunit; hence, we name this step k(gamma). Moreover, the only model that is consistent with our data and many other observations in the literature suggests that reversible hydrolysis/synthesis can only occur in the active site of the beta(TP) conformer (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628).     

Rate of chase-promoted hydrolysis of ATP in the high affinity catalytic site of beef heart mitochondrial ATPase

Incubation of [gamma-32P]ATP with a molar excess of the soluble, homogeneous ATPase from beef heart mitochondria (F1) results in binding of substrate primarily in a single, very high affinity (KA = 10(12) M-1) catalytic site and in a slow rate of hydrolysis characteristic of single site catalysis. Subsequent addition of millimolar concentrations of nonradioactive ATP as a cold chase, sufficient to fill catalytic sites on the enzyme, results in an acceleration of hydrolysis of bound radioactive ATP of as much as 10(6)-fold, that is, to Vmax rates (Cross, R.L., Grubmeyer, C., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12101-12105). For this reason, it was proposed that the high affinity catalytic site is a normal catalytic site on the molecule. Recently, Bullough et al. (Bullough, D.A., Verburg, J.G., Yoshida, M., and Allison, W.A. (1987) J. Biol. Chem. 262, 11675-11683) reported that when 5 to 20 microM concentrations of nonradioactive ATP were added as a cold chase to an enzyme-substrate complex consisting of F1 and ATP bound to the high affinity catalytic site, hydrolysis of the chase was commensurate with the turnover rate of the enzyme, whereas the hydrolysis of bound ATP was considerably slower. These authors suggested that the high affinity catalytic site on F1 is not a normal catalytic site. This paper shows, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to Vmax rates following addition of 5 microM ATP as a cold chase. Hydrolysis of bound ATP appears to precede that of the chase. The weight of the available evidence continues to support the original suggestion that the high affinity catalytic site of beef heart F1 is a normal catalytic site.     

Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1

A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1). The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed. The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores. The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R. rubrum Fo-F1 complex. The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin. In contrast, chromatophores reconstituted with the native R. rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin. These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes. They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form.     

Thermodynamics of hydrolysis of oligosaccharides

Microcalorimetry has been used to determine enthalpy changes for the hydrolysis of a series of oligosaccharides. High-pressure liquid chromatography was used to determine the extents of reaction and to check for any possible side reactions. The enzyme glucan 1,4-alpha-glucosidase was used to bring about the following hydrolysis reactions: (A) maltose(aq) + H2O(liq) = 2D-glucose(aq); (B) maltotriose(aq) + 2H2O(liq) = 3D-glucose(aq); (C) maltotetraose(aq) + 3H2O(liq) = 4D-glucose(aq); (D) maltopentaose(aq) + 4H2O(liq) = 5D-glucose(aq); (E) maltohexaose(aq) + 5H2O(liq) = 6D-glucose(aq); (F) maltoheptaose(aq) + 6H2O(liq) = 7D-glucose(aq); (G) amylose(aq) + nH2O(liq) = (n + 1) D-glucose(aq); and (H) panose(aq) + 2H2O(liq) = 3D-glucose(aq); (J) isomaltotriose(aq) + 2H2O(liq) = 3D-glucose(aq). The enzyme beta-fructofuranosidase was used for the reactions: (K) raffinose(aq) + H2O(liq) = alpha-D-melibiose(aq) + D-fructose(aq); and (L) stachyose(aq) + H2O(liq) = o-alpha-D-galactopyranosyl-(1----6)- alpha-o-D-galactopyranosyl-(1----6)-alpha-D-glucopyranose + D-fructose(aq). The results of the calorimetric measurements (298.15 K, 0.1 M sodium acetate buffer, pH 4.44-6.00) are: delta H0A = -4.55 +/- 0.10, delta H0B = -9.03 +/- 0.10, delta H0C = -13.79 +/- 0.15, delta H0D = -18.12 +/- 0.10, delta H0E = -22.40 +/- 0.15, delta H0F = -26.81 +/- 0.20, delta H0H = 1.46 +/- 0.40, delta H0J = 11.4 +/- 2.0, delta H0K = -15.25 +/- 0.20, and delta H0L = -14.93 +/- 0.20 kJ mol-1. The enthalpies of hydrolysis of two different samples of amylose were 1062 +/- 20 and 2719 +/- 100 kJ mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)