Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

The catalytic metal atoms of cobalt substituted liver alcohol dehydrogenase.

The catalytic and non-catalytic Zn atom pairs of horse liver alcohol dehydrogenase (LADH) have been replaced sequentially either by ⁶⁵Zn, Co or ⁶⁵Zn and Co. The Co derivatives exhibit characteristic spectra. When Co replaces the Zn atoms which exchange secondly, enzymatic activity is altered, and both imidazole and 1,10-phenanthroline (OP) significantly modify the spectrum of the catalytic Co atoms. Further, due to the removal of cobalt, the instantaneous and reversible OP inhibition of the native enzyme becomes time-dependent and irreversible. Jointly, these data identify the pair of metal atoms of LADH which exchange secondly under the present conditions as the catalytic one. The approach described provides a basis for the differentiation of catalytic and non-catalytic metal atoms of multichain metalloenzymes.     

Cobalt(II)-substituted class III alcohol and sorbitol dehydrogenases from human liver

The catalytic zinc atoms in class III (chi) alcohol dehydrogenase (ADH) and sorbitol dehydrogenase (SDH) from human liver have been specifically removed and replaced by cobalt(II) with a new ultrafiltration technique. The electronic absorption spectrum of class III cobalt ADH (epsiolon 638 = 870 M-1 cm-1) is nearly identical with those of active site substituted horse EE and human class I (beta 1 beta 1) cobalt ADH. Thus, the coordination environment of the catalytic metal is strictly conserved in these enzymes. However, significant differences are noted when the spectra of class III ADH-coenzyme complexes are compared to the corresponding spectra of the horse enzyme. The spectrum of class III ADH.NADH is split into three bands, centered at 680, 638, and 562 nm. The class III ADH.NAD+ species resembles the alkaline form of the corresponding horse enzyme complex but without exhibiting the pH dependence of the latter. These spectral changes underscore the role of the coenzymes in differentially fine tuning the catalytic metal for its particular function in each ADH. The noncatalytic zinc of class III ADH exchanges with cobalt at pH 7.0. While 9 residues out of 15 in the loop surrounding the noncatalytic zinc of class III ADH differ from those of the class I ADH, the electronic absorption spectra of cobalt in the noncatalytic metal site of class III ADH establish that the coordination environment of this site is conserved as well. The spectrum of cobalt SDH differs significantly from those of cobalt ADHs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal stoichiometry, coenzyme binding, and zinc and cobalt enchange in highly purified yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase, purified from baker's yeast under conditions which exclude contamination by extraneous metal ions, is homogeneous by analytical ultracentrifugation and disc gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a molecular weight of 149,000 as determined by ultracentrifugation time-lapse photography and exhibits specific activities of 430 to 480 U/mg. Zinc analysis by three independent, highly sensitive methods, i.e., atomic absorption spectrometry, atomic fluorescence spectrometry, and microwave-induced plasma emission spectrometry, demonstrates 4 g-atom of catalytically essential Zn per mole of enzyme. No other metal atoms are present in stoichiometrically significant quantities as assessed by emission spectrography. The Stoichiometry of coenzyme binding, 4 mol of NADH/mol of enzyme, is identical to that of zinc, consistent with one coenzyme binding site and one zinc atom per enzyme subunit. Conditions for exchange of the four catalytically essential zinc atoms with ⁶⁵Zn have been developed. These atoms exchange identically under all conditions examined. The resultant radiolabeled enzyme, l(YADH)⁶⁵Zn₄], has the same metal content, specific enzymatic activity, and coenzyme binding properties as the native enzyme. The ⁶⁵Zn of this enzyme serves to monitor the extent and site specificity of cobalt replacement. The fully cobalt-substituted enzyme, [(YADH)Co₄], has a specific activity of 80 U/mg, 17% that of the Zn enzyme, and exhibits absorption and circular dichroic spectra which are consistent with coordination by one or more sulfur ligands in a distorted tetrahedral geometry.     

Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernl?hr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.     

Differential expression of ribulose-1,5-bisphosphate carboxylase in reciprocal F1 hybrids of a C3 and a C4-likeFlaveria species

Stable reciprocal hybrids between Flaveria pringlei (C3) and F. brownii (C4-like) have been produced by standard breeding techniques. There are no differences in the isoelectric focusing patterns of the catalytic subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase from F. pringlei, F. brownii, or the reciprocal hybrids. The enzyme from both species also contains an identical noncatalytic subunit polypeptide. However, the carboxylase enzyme from F. brownii contains another isomeric form of noncatalytic subunit polypeptide which is resolveable by isoelectric focusing. This isomeric form constitutes about 50% of the total noncatalytic subunits in this species. It comprises only about 10% of the total noncatalytic subunit population in the C3 x C4 plants, but about 42% of the noncatalytic subunits in the reciprocal cross. The concentrations of the holoenzyme in the reciprocal hybrids are comparable to those of the respective maternal parent. We hypothesize that a differential inheritance of parental chloroplasts by the reciprocal hybrids may be associated with this apparent maternal influence on the expression of the noncatalytic polypeptides and the holoenzyme concentration.     

Horse liver alcohol dehydrogenase derivatives containing nickel(II) and cobalt(lI) in the noncatalytic metal binding site

The noncatalytic zinc in horse liver alcohol dehydrogenase was selectively replaced by nickel(II). This novel species, Zn(c)₂Ni(n)₂⁴ horse liver alcohol dehydrogenase (where c denotes the catalytic and n denotes the noncatalytic site) was compared to Zn(c)₂Co(n)₂ horse liver alcohol dehydrogenase with respect to its absorption, circular dichroism and magnetic circular dichroism spectra, as well as its magnetic moment. For Zn(c)₂Co(n)₂ horse liver alcohol dehydrogenase (prepared according to refs. 1 and 2) the extinction coefficients were redetermined in the UV, visible and near-infrared region and the molar ellipticities in the range 300-800 nm. The average magnetic moment was determined by the NMR method as 4.5-5.0 B.M. The results confirm a tetrahedral structure in the zinc-cobalt enzyme. In contrast, the spectroscopic data and the zero magnetic moment support a planar geometry for the nickel(Il) bound in the noncatalytic site. Zn(c)₂Ni(n)₂ horse liver alcohol dehydrogenase is very temperature-sensitive and precipitates after short exposure to room temperature. Stored in the cold it has the same activity as the native enzyme. The results indicate that the protein is flexible in the loop region binding the noncatalytic metal ion and that it may retain catalytic activity even in a partially distorted conformation.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Structure of the prolidase from Pyrococcus furiosus

The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781-9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of two functionally related enzymes, aminopeptidase P (APPro) and creatinase. In addition, the catalytic C-terminal domain of Pfprol has a polypeptide fold resembling that of the sole domain of a fourth enzyme, methionine aminopeptidase (MetAP). The active sites of APPro and MetAP, like that of Pfprol, include a dinuclear metal center. The metal ligands in the three enzymes are homologous. Comparisons with the molecular structures of APPro and MetAP suggest how Pfprol discriminates against oligopeptides and in favor of Xaa-Pro substrates. The crystal structure of Pfprol was solved by multiple-wavelength anomalous dispersion. The crystals yielded diffraction data of relatively high quality and resolution, despite the fact that one of the two protein subunits in the asymmetric unit was found to be significantly disordered. The final R and R(free) values are 0.24 and 0.28, respectively.     

The intrinsic zinc atoms of yeast alcohol dehydrogenase

The intrinsic Zn content of yeast alcohol dehydrogenase (YADH) has been determined by three highly sensitive analytical techniques. The enzyme prepared from baker's yeast has a specific activity of 430–460 U/mg and contains 4 intrinsically bound Zn atoms per tetrameric enzyme of molecular weight 150,000. The enzyme is homogeneous by disc gel electrophoresis and analytical ultracentrifugation and remains stable and fully active on prolonged storage. YADH samples from commercial sources, while of high activity, can initially contain more than 4 g-atom of Zn/mole, but dialysis against EDTA removes these adventitious Zn atoms which do not bear a consistent relationship to enzymatic activity, in accord with earlier investigations. Apparently, they are bound to the enzyme in a manner different from that of the catalytically essential Zn atoms and likely represent contamination. The 4 intrinsic Zn atoms exchange fully with ⁶⁵Zn(II) resulting in [(YADH)⁶⁵Zn₄] which exhibits the same specific activity and stability as the native enzyme.     

Metal specificity is correlated with two crucial active site residues in Escherichia coli alkaline phosphatase

Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymes can use cobalt for catalysis. To understand the structural basis for the altered metal specificity and the ability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of the inactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328W enzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in the strength of the interaction with the product phosphate (P(i)). Since release of P(i) is the slow step in the mechanism at alkaline pH, the enhanced binding of P(i) in the WT_Co structure explains the observed decrease in activity, while the weakened binding of P(i) in the HW_Co structure explains the observed increase in activity. These alterations in P(i) affinity are directly related to alterations in the coordination of the metals in the active site of the enzyme.     

Reactivation of apo horse liver alcohol dehydrogenase with the monovalent metal ion Ag(I)

Each subunit of the liver alcohol dehydrogenase dimer contains one catalytic and one structural Zn(II) atom. Enzyme with the catalytic metal atoms selectively removed is inactive but can be partly reactivated in the presence of Ag(I) ions. Reactivation results from Ag(1) ions entering the empty metal-binding site in the catalytic center. The specific activity of this silver enzyme reached 24% of the native enzyme. Atomic absorption analysis gave equal amounts of Ag(I) and Zn(II), corresponding to one mole of each metal per monomer. Metal-directed affinity labelling using bromo-imidazolyl propionate showed that the properties of the silver-reactivated enzyme were distinct from those of the native enzyme.     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

Heterogeneity in the rapidly exchanging metals of horse liver alcohol dehydrogenase.

Substitution of the two rapidly exchanging zinc atoms of liver alcohol dehydrogenase by cobalt is biphasic; replacement by the first cobalt occurs at a rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15 minutes}$) approximately ten times faster than substitution by the second cobalt atom. The hybrid enzyme containing one gram atom of cobalt has a characteristic visible absorption spectrum which is not perturbed by NADH or 1,10-phenanthroline. The fluorescence of NADH or ε-NAD bound to the hybrid is not quenched. These data indicate a previously unrecognized heterogeneity in the rapidly exchanging zinc atoms; one of the exchange labile zinc atoms is located at a structural metal binding site rather than an active site.     

Magnesium(II) and zinc(II)-protoporphyrin IX's stabilize the lowest oxygen affinity state of human hemoglobin even more strongly than deoxyheme.

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha2(Fe)beta2(M) and alpha2(M)beta2(Fe); M=Mg(II), Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha2(Fe)beta2(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effectors, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha2(Fe)beta2(Mg) and alpha2(Fe-CO)beta2(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effectors is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution.We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins.     

Metallation of the transition-state inhibitor N-methyl mesoporphyrin by ferrochelatase: implications for the catalytic reaction mechanism

Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.     

2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase

The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.     

Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-beta-lactamase catalyzed hydrolysis of beta-lactams

Metallo-beta-lactamases are native zinc enzymes that catalyze the hydrolysis of beta-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted beta-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pK(a) = 8.9 +/- 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H(2)O/D(2)O) of 5.2 +/- 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt beta-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pK(a) 6.32 +/- 0.1 and 7.47 +/- 0.1 being in their deprotonated basic forms and one group of pK(a) 9.48 +/- 0.1 being in its protonated form.     

Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides

When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.