Active Site Zn Research Articles

Inhibition of metallo-β-lactamases by a series of thiol ester derivatives of mercaptophenylacetic acid

A series of mercaptophenylacetic acid thiol esters bearing a phenyl substituent adjacent to the carboxylic acid function has been shown to be inhibitors of metallo-β-lactamases. The inhibition of the Bacteroides fragilis CfiA and Bacillus cereus II metallo-β-lactamases was Zn 2+ dependent, greater inhibition being observed at 1 μM ZnSO 4 than at 100 μM ZnSO 4. Despite this Zn 2+ dependency, isothermal titration calorimetry studies illustrated that representative compounds had no detectable affinity for Zn 2+ ( K>1 mM). This indicates that their mode of inhibition was not by chelation of the active site Zn 2+. Greatest potency was observed against the Stenotrophomonas maltophilia L1 metallo-β-lactamase with I 50 values of between <1.95 μM and 6 μM and SB-217843 exhibited a similar level of inhibition of this enzyme at 1 and 100 μM Zn 2+ (I 50 values 5 and 6 μM, respectively). Inhibition of B. cereus II metallo-β-lactamase by SB-218018 and SB-217782 was competitive with K i values of 185 μM and 1500 μM, respectively. Therefore, these compounds are specific inhibitors of metallo-β-lactamases and provide further probes of the active sites of these enzymes.

Kinetic characterization of the serralysins: a divergent catalytic mechanism pertaining to astacin-type metalloproteases.

Substrates HO2CCH2CH2CO- and HOCH2CHOHCHOHCO-Phe-Leu-Ala-5-nitro-2-pyridinamide are cleaved efficiently at the acylarenamide linkage, with a convenient spectrophotometric assay, by the Serratia and Pseudomonas approximately 50-kDa extracellular metalloproteases (serralysins). The pH range of catalytic activity extends uniformly from 4 to greater than 10 (k(cat)/Km approximately 10(3) s(-1) M(-1), similar profile for k(cat)). Substrate analogue hydroxamic acid Cbz-Leu-Ala-NHOH competitively inhibits serralysin (Ki 0.04 mM), with a pH dependence indicating that either a displaced metal-bound H2O or a similarly motile enzymic phenol residue (Tyr216) that is crystallographically found ligated to the active-site Zn2+ of the uncomplexed enzyme must have a pKa of approximately 5. A chemical catalytic mechanism of proteolysis consistent with the kinetic data is proposed, in which Tyr216-ArO-, in the course of being released from the active-site metal ion, deprotonates a water molecule attacking the Zn2+-activated substrate linkage, leading to a metal-coordinated tetrahedral oxyanion adduct that subsequently fragments.

Molecular Cloning of Acetone Cyanohydrin Lyase from Flax (Linum usitatissimum): DEFINITION OF A NOVEL CLASS OF HYDROXYNITRILE LYASES

Acetone cyanohydrin lyase from Linum usitatissimum is a hydroxynitrile lyase (HNL) which is involved in the catabolism of cyanogenic glycosides in young seedlings of flax. We have isolated a full-length cDNA clone encoding L. usitatissimum HNL (LuHNL) from a cDNA expression library by immunoscreening. LuHNL cDNA was expressed in Escherichia coli and isolated from the respective soluble fraction in an active form which was biochemically indistinguishable from the natural enzyme. An open reading frame of 1266 base pairs encodes for a protein of 45,780 kDa. The derived amino acid sequence shows no overall homologies to the to date cloned HNLs, but has significant similarities to members of the alcohol dehydrogenase (ADH) family of enzymes. In particular, the cysteine and histidine residues responsible for coordination of an active site Zn2+ and a second structurally important Zn2+ in alcohol dehydrogenases are conserved. Nevertheless, we found neither alcohol dehydrogenase activity in LuHNL nor HNL activity in ADH. Moreover, well known inhibitors of ADHs, which interfere with the coordination of the active site Zn2+, fail to affect HNL activity of LuHNL, suggesting principally different mechanisms of cyanohydrin cleavage and alcohol oxidation. Interestingly, LuHNL like ADH and Prunus serotina (PsHNL) possesses an ADP-binding betaalphabeta unit motif, pointing to the possibility that the non-flavoprotein PsHNL and the flavoprotein LuHNL have developed from two independent lines of evolution of a common ancestor with an ADP-binding betaalphabeta unit.

Is cyanate a carbonic anhydrase substrate?

A study was undertaken to investigate whether diverse carbonic anhydrase (CA) isozymes (both native Zn as well as cobalt-substituted) are able to catalyze the hydrolysis of anions such as cyanide, cyanate, and thiocyanate. A controversy exists between the crystallographic and spectroscopic data of CA II-anion adducts. In the former case it has been shown that "metal poisons" such as CN- and CNO- are not directly coordinated to the active site Zn(II) ion whereas spectroscopic studies indicate otherwise. A theoretical study in the above systems did not resolve this controversy, since it was calculated that all three anions can act as CA substrates. In this paper we prove experimentally that none of them may act as substrates of CA and propose an explanation to the above controversy, discussing the mode of binding of small molecules within the enzyme active site.

Glutathione-independent formaldehyde dehydrogenase from Pseudomons putida: survey of functional groups with special regard for cysteine residues.

The role of cysteine residues for structure and function of formaldehyde dehydrogenase from Pseudomonas putida was analysed by amino acid sequence comparison, homology-based structure modeling, site-directed mutagenesis, and chemical modification. Five out of seven cysteine residues found in the enzyme were concluded to coordinate with an active site zinc (Cys-46) and structural zinc atoms (Cys-97, -100, -103, and -111) from the sequence comparison with other Zn-containing medium-chain alcohol dehydrogenase homologues. The three-dimensional structure model based on the known structure of the horse liver E-type alcohol dehydrogenase (ADH) indicated that Cys-257 is located very far from the active site Zn and NAD+ binding region, suggesting that Cys-257 does not participate in the enzyme reaction. The structure also suggested that Cys-166 does not coordinate to active site Zn, but Asp-169 functions as a Zn-ligand, instead.

Mechanism of aldehyde oxidation catalyzed by horse liver alcohol dehydrogenase.

The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed by horse liver alcohol dehydrogenase (HLADH) has been investigated using the HLADH structure at 2.1 A resolution with NAD+ and pentafluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations with HLADH were obtained by a best-fit superimposition of benzaldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II)ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(II) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initio-derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21,40-56]. Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MD1) enzyme with NAD+, benzaldehyde, and zinc-ligated HO-in the active site; (MD2) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxygen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempirical PM3 calculations. Using PM3, model systems were calculated of ground states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc-bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resided at a distance of 2.8-5.5 A from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 transition state for attack of the Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, which has an O...C distance of 1.877 A. HLADH catalysis of the aldehyde hydration would require very little motion aside from that in the ground state. Two simulations of benzaldehyde hydrate ligated to zinc (MD2 and MD3) both showed close approach of the aldehyde hydrate hydrogen to NAD+C4, varying from 2.3 to 3.3 A, seemingly favorable for the hydride transfer reaction. The MD2 configuration does not allow proton shuttling. On the other hand, when the pro-S oxygen is ligated to zinc (MD3), the proton on the pro-R oxygen averages 2.09 A from the hydroxyl oxygen of Ser48 such that initiation of shuttling of protons via Ser48 to the ribose 2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen is sterically favorable. PM3 calculations suggest that this proton shuttle represents a stepwise reaction which occurs subsequent to hydride transfer. The PM3 transition state for hydride transfer based on the MD3 configuration has the transferring hydride 1.476 A from C4 of NAD+ and 1.433 A from the aldehyde alpha-carbon.

Refined structure of Cu-substituted alcohol dehydrogenase at 2.1 A resolution.

Liver alcohol dehydrogenase (LADH) is a Zn(II)-dependent dimeric enzyme. LADH with the active-site Zn(II) substituted by Cu(II) resembles blue (type I) copper proteins by its spectroscopic characteristics. In this work we present the X-ray structure of the active site Cu(II)-substituted LADH complex with NADH and dimethyl sulfoxide (DMSO). The structure was solved by molecular replacement. The space group is P2(1) with cell dimensions a = 44.4, b = 180.6, c = 50.8 A and beta = 108 degrees. There is one dimer of the enzyme in the asymmetric unit. The refinement was carried out to a crystallographic R-factor of 16.1% for 41 119 unique reflections in the resolution range 12.0 to 2.1 A. The coordination geometry of Cu(II) in LADH is compared with the active-site metal coordination in the Zn-LADH-NADH-DMSO complex and blue-copper proteins. The distances from the metal to the protein ligands (Cys46, His67 and Cys174) are similar for the Zn(II) and Cu(II) ions. The distances of the O atom of the inhibitor DMSO to the Cu(II) ion in the two subunits of the dimer are 3.19 and 3.45 A. These are considerably longer than the corresponding distances for the Zn(II) enzyme, 2.19 and 2.15 A. The Cu(II) ion is positioned nearly in the plane of the three protein ligands (NS(2)) with a geometry similar to the trigonal arrangement of the three strongly bound ligands (N(2)S) in blue-copper proteins. This coordination probably accounts for the similarity of the spectral characteristics of Cu(II)-LADH and type I copper proteins.

Activation and Characterization of Procarboxypeptidase B from Human Plasma

Recently we reported the isolation and cloning of a novel plasma procarboxypeptidase B that binds plasminogen [Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., & Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838]. This plasma procarboxypeptidase is structurally similar to tissue procarboxypeptidases, and initial substrate studies showed that this plasma protein behaves like a basic carboxypeptidase and is now known as human plasma procarboxypeptidase B (pro-pCPB). However, unlike the tissue procarboxypeptidases, pro-pCPB is extremely unstable to trypsin activation. Trypsin cleaves pro-pCPB at two sites: Arg-92 and Arg-330. Cleavage at Arg-92 releases the activation peptide and generates an active enzyme. However, cleavage at Arg-330 inactivates pCPB. This renders the characterization of pCPB difficult. We have found that 6-amino-n-hexanoic acid (epsilon ACA), a compeptitive inhibitor of basic carboxypeptidases, selectively limits trypsin cleavage of pro-pCPB. In the presence of epsilon ACA, trypsin cleavage at Arg-330 is significantly limited while the cleavage at Arg-92 is unaffected. Using this approach, active pCPB can now be obtained. Kinetic characterization shows that pCPB behaves like other known basic carboxypeptidases. pCPB is more specific for substrates with C-terminal arginine than those with C-terminal lysine for all the natural and synthetic peptides tested. It also hydrolyzes the synthetic ester substrate more efficiently than the synthetic peptide substrate, especially at high pH. The active site Zn2+ can be replaced with other metals with change in substrate specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis.

Competitive inhibition as a function of pH for the metalloendoprotease thermolysin by derivatives of L-alpha-(2-hydroxyphenyl)benzenepropanoyl-L- tryptophanylglycylglycine exhibits a diagnostic bell shape. Binding is maximal between two pKa values: on the acidic limb the apparent Ki value is regulated by an unchanging enzymic ionization (pKa 5.3) which is also seen in the substrate-hydrolysis kinetics (kcat/Km), whereas the alkaline limb for inhibition varies and depends specifically on the pKa of the phenolic group in the inhibitor. Although it should be the phenolate form of the inhibitor that co-ordinates more efficiently to the active-site Zn2+, the apparent Ki shifts from pH-independent at pH values immediately below the inhibitor's pKa to progressively weaker binding at higher pH. This is explained by an anomalous acidity for the exchangeable solvent molecule that is attached to enzymic Zn2+ in the absence of substrate or inhibitor. Since OH- cannot be displaced from the enzyme as readily as H2O, a compensating pKa of 5.3 possessed by Zn(2+)-bound water rationalizes the binding characteristics, yielding the level pH profile exhibited at intermediate pH values. Recognition of the implicit heightened Lewis acidity of the metal ion in thermolysin leads to a revision of the mechanism of catalysis. The substrate amide bond becomes activated for hydrolysis by carbonyl-group co-ordination to the especially acidic Zn2+ ion (completely displacing the H2O/OH- species otherwise bound). The imidazole group of enzymic residue His-231, also discerned in the pH profile for kcat/Km from its pKa of 8, provides general-base assistance for hydration of the activated scissile linkage in the first committed step of catalysis. Additional evidence from inhibition patterns shows how substrate-binding energy may be employed in this scheme to promote hydrolysis of peptides by thermolysin.

Acid-base catalytic mechanism of dihydropyrimidinase from pH studies

The pH dependence of kinetic parameters and solvent deuterium isotope effects have been used to probe the mechanism of the dihydropyrimidinase from the liver of pig and calf. The V/K for 5,6-dihydrouracil (DHU) (or the alternative substrate glutarimide) measured with either the native zinc or cadmium-substituted enzymes decreases at both low and high pH giving pK values of about 7.5-8 and 9-10. The low pK value observed in V is perturbed significantly to lower pH (approximately 6), and the high pK is not observed. The binding of glutarate monoamide is optimum when the group with a pK of 7.7 is protonated, and this same group must be protonated for the reverse reaction, that is, formation of DHU from N-carbamoyl-beta-alanine. These data are consistent with a general base mechanism and in addition suggest that the enzyme is present initially with a water bound to the active site zinc. The enzymic general base with a pK of 7.5-8 is required to activate water for nucleophilic attack on the C-4 of 5,6-dihydrouracil which is directly coordinated to the active site zinc. The second group with a pK of 9-10 likely reflects Zn-water ionization of the free enzyme. The water bound to the active site Zn is displaced by reactant binding, and thus the pK of 9-10 is not observed in the V profile. Solvent deuterium isotope effects are near unity on the V/K for the natural substrate 5,6-dihydrouracil, but a finite effect of 1.6 is observed on V.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereo-specific inhibition of sea urchin envelysin (hatching enzyme) by a synthetic autoinhibitor peptide with a cysteine-switch consensus sequence

Inhibition of envelysin, a metalloproteinase which dissolves the fertilization envelope of sea urchin embryo, was studied using a synthetic autoinhibitor peptide, Ac-Pro-Arg-Cys-Gly-Val-Pro-Asp-Val-NH 2, with a ‘cysteine-switch’ consensus sequence. Although its effect is reversible, the hatching of sea urchin embryos was effectively delayed by 0.5 mM of the peptide. When α 1-proteinase inhibitor was used as the substrate, envelysin was inhibited by the autoinhibitor and an Ala 6 analogue, but not by a d-Cys 3 analogue. However, envelysin was weakly inhibited by both d- and l-cysteines to the same extent. Snake venom α-protease exhibited cleavage and inhibition behavior similar to envelysin with a little weaker stereo-specificity. The results suggest that the coordination of the autoinhibitor Cys residue with the envelysin active site Zn is established only after the amino acid residues on both sides of the Cys residue get into an appropriate interaction with the catalytic site residues, and that the precise orientation of the cysteine SH group is essential. By contrast, thermolysin was weakly inhibited by the three peptide non-stereo-specifically. Furthermore, thermolysin cleaved the autoinhibitor at the Cys 3 Gly 4 bond when incubated without substrate.

Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase.

Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.

Raman spectral evidence for an anhydride intermediate in the catalysis of ester hydrolysis by carboxypeptidase A

Single crystals of carboxypeptidase A, grown at pH 7.5, have been soaked with solutions of a chromophoric substrate, the ester of p-dimethylaminobenzoic acid and L-β-phenyllactic acid, at pH=8. The enzyme in the crystal catalyzes the conversion of the ester to the acid and alcohol products. During this process, pre-resonance-enhanced Raman spectra of the interior of the crystal were taken using a laser Raman microscope. In addition to the Raman bands characteristic of the substrate and the acid product, weak but reproducible bands are observed in the frequency region of 1700-1800 cm −1 . Bands in this region are unique to organic acid anhydrides and indicate the accumulation of an anhydride intermediate during catalysis. A comparison of these spectra with those of model compounds designed to mimic a pure anhydride intermediate and a Zn(II)-complexed anhydride intermediate suggests the active-site Zn(II) is sometimes bound to the carbonyls of the anhydride intermediate

Inhibition of leukotriene A4 hydrolase/aminopeptidase by captopril.

Captopril ((2S)-1-(3-mercapto-2-methyl-propionyl)-L-proline) inhibited the bifunctional, Zn(2+)-containing enzyme leukotriene A4 hydrolase/aminopeptidase reversibly and competitively with Ki = 6.0 microM for leukotriene B4 formation and Ki = 60 nM for L-lysine-p-nitroanilide hydrolysis at pH 8. Inhibition was independent of pH between pH 7 and 8, the optimum range for each catalytic activity. Half-maximal inhibition of leukotriene B4 formation by intact erythrocytes and neutrophils required 50 and 88 microM captopril, respectively. In neutrophils and platelets neither 5(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroxyeicosatetraenoic acid, nor leukotriene C4 formation were reduced, indicating selective inhibition of leukotriene A4 hydrolase/aminopeptidase, not 5-lipoxygenase, 12-lipoxygenase, or leukotriene C4 synthase. In whole blood, captopril inhibited leukotriene B4 formation with an accompanying redistribution of substrate toward formation of cysteinyl leukotrienes. The decrease in leukotriene B4 was more substantial than the corresponding increase in cysteinyl leukotrienes suggesting that nonenzymatic hydration predominates over transcellular metabolism of leukotriene A4 by platelets during selective inhibition of leukotriene A4 hydrolase. Enalapril dicarboxylic acid and Glu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro which inhibit angiotensin-converting enzyme: angiotensin I, bradykinin, and N-[3-(2-furyl)acryloyl]Phe-Gly-Gly which are substrates; and chloride ions which activate angiotensin-converting enzyme did not modulate leukotriene A4 hydrolase/aminopeptidase activity. The results indicate that: (i) the sulfhydryl group of captopril is an important determinant for inhibition of leukotriene A4 hydrolase/aminopeptidase, probably by binding to an active site Zn2+; (ii) aminopeptidase and leukotriene A4 hydrolase display differential susceptibility to inhibition; (iii) there is minimal functional similarity between angiotensin-converting enzyme (peptidyl dipeptidase) and leukotriene A4 hydrolase/aminopeptidase; (iv) captopril may be a useful prototype to identify more potent and selective leukotriene A4 hydrolase inhibitors.

Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II)

Treatment of Escherichia coli dihydroorotase (a homodimer of subunit molecular weight 38,729) containing only the 1 active site Zn(II) ion per subunit with the sulfhydryl reagent N-(ethyl)-maleimide (NEM) blocks the two external Zn(II) sites per subunit and dramatically lessens the precipitation caused by high concentrations of Zn(II); stabilizes the enzyme partially against air oxidation and dilution inactivation; makes the active site Zn(II) easier to remove; and lowers Km and increases kcat. Treatment of NEM-blocked dihydroorotase ((NEM)dihydroorotase) with the chelator 2,6-pyridinedicarboxylic acid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(II) with a half-life of 15 min, allowing the production of milligram amounts of moderately stable apo-(NEM)dihydroorotase in about 80% yield. Treatment of apo-(NEM)dihydroorotase with Co(II) at pH 7.0 produces (NEM)dihydroorotase completely substituted at the active site with Co(II) in 100% yield: analysis gives 0.95-1.1 g atoms of Co(II) per active site and 0.03-0.05 g atoms of Zn(II) per active site. This Co(II)-(NEM)dihydroorotase is hyperactive at pH 8. The electronic absorption spectrum of Co(II)-(NEM)dihydroorotase at pH 6.5 implicates an active site thiol group as a ligand to the metal ion. The spectrum is inconsistent with tetrahedral coordination of the active site metal ion and is most consistent with a pentacoordinate structure.

Production, purification and spectral properties of metal-dependent β-lactamases of Bacillus cereus

New methods for the production of consistently high levels of metal-dependent beta-lactamases (beta-lactamhydrolase, EC 3.5.2.6) from strains 569/H/9 and 5/B/6 of Bacillus cereus are described which have significant advantages over those reported previously. For example, these techniques do not require a fermentor with pH-stat capabilities. We also describe rapid very-high-yield purification schemes for the metal-dependent beta-lactamases from these strains, employing high-performance ultrafiltration (HPUF) and mass ion exchange techniques. Furthermore, we have developed improved methods for the removal of the active site Zn(II) and reconstitution of the beta-lactamase enzymatic activity with Co(II), which result in higher recovery of the original activity than previously reported. In order to characterize the purified beta-lactamases II of B. cereus 569/H/9 and 5/B/6 we have examined the molecular weights, and steady state kinetic parameters of Zn(II) enzymes, and the electronic and EPR spectra of the Co(II)-reconstituted enzymes. EPR spectra of CO(II)-reconstituted beta-lactamase from B. cereus 5/B/6 have not been previously reported.

Ionization states of the complex formed between 2-benzyl-3-phosphonopropionic acid and carboxypeptidase A.

The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.

Preparation and reconstitution with divalent metal ions of class I and class II Clostridium histolyticum apocollagenases.

Both gamma- and zeta-collagenases from Clostridium histolyticum are fully and reversibly inhibited by 1,10-phenanthroline at pH 7.5 in the presence of 10 mM CaCl2 with KI values of 0.11 and 0.040 mM, respectively. The inhibition is caused by removal of the single, active-site Zn(II) present in each of these enzymes. The nonchelating analogue 1,5-phenanthroline has no effect on the activity of either enzyme. Dialysis of the enzymes in the presence of 1,10-phenanthroline, followed by back dialysis against buffer containing no chelating agent, gives the respective apocollagenases. Both apoenzymes can be instantaneously and fully reactivated by the addition of 1 equiv of Zn(II). Variable amounts of activity are restored to both apocollagenases by Co(II) and Ni(II) and to gamma-apocollagenase by Cu(II). The activity titration curve for gamma-apocollagenase with Co(II) and Scatchard plots for the reconstitution of gamma-apocollagenase with Cu(II) and Ni(II) and of zeta-apocollagenase with Ni(II) and Co(II) indicate that all activity changes are the result of binding of a single equivalent of these divalent metal ions at the active site of the collagenases. Cd(II) and Hg(II) do not restore measurable activity to either apoenzyme.

Neutral metal-bound water is the base catalyst in liver alcohol dehydrogenase.

The catalytic role of the active site metal-water complex in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) is investigated on the basis of a comparative analysis of the pH dependence of steady-state kinetic parameters of the native and active-site-specific Co2+-reconstituted enzyme and on the basis of assignment of the coordination environment of the Co2+ by electron paramagnetic resonance methods. The pH dependence of the kinetic parameters for the oxidation of benzyl alcohol reveals two ionizations (pK1 approximately equal to 6.7; pK2 approximately equal to 10.6) that govern kcat and belong to the ternary enzyme-NAD+-alcohol complex and two ionizations (pK1' approximately equal to 7.5; pK2' approximately equal to 8.9) that govern kcat/Km and belong to the binary enzyme-NAD+ complex. The ionizations pK2 and pK2' decrease by 0.5-1 pK alpha unit upon replacement of the active site Zn2+ by Co2+. A similar metal ion dependence of pK2 and pK2' is observed for the oxidation of 2-propanol. We attribute these ionizations to a metal-bound water molecule. The zero-field splitting energy of the Co2+ in the binary enzyme-NADH complex and the ternary enzyme-NADH-CF3CH2OH complex is approximately equal to 22 cm-1, indicative of a pentacoordinate species. Binding of a water molecule to the metal ion as the fifth ligand in the ternary enzyme-NADH-CF3CH2OH complex is confirmed on the basis of magnetic interactions of H2(17)O with Co2+. The results indicate that the active site metal ion in catalytically competent ternary enzyme-coenzyme-substrate complexes is pentacoordinate and is ligated by a neutral water molecule in the physiological pH range. We suggest that the neutral metal-bound water molecule serves as the base catalyst for proton abstraction in alcohol oxidation.

Determination and analysis of the 2 Å structure of copper, zinc superoxide dismutase

The structure of bovine erythrocyte Cu, Zn superoxide dismutase has been determined to 2 Å resolution using only the larger structure factors beyond 4 Å. The enzyme crystallizes in space group C2 with two dimeric enzyme molecules per asymmetric unit. All four crystallographically independent subunits were fitted separately to the electron density map at 2 Å resolution on the University of North Carolina GRIP-75 molecular graphics system. Atomic co-ordinates were refined using the Hendrickson & Konnert (1980) program for stereochemically restrained refinement against structure factors, which allowed the use of non-crystallographic symmetry. The crystallographic residual error for the refined model was 25.5% with a root-mean-square deviation of 0.03 Å from ideal bond lengths and an average atomic temperature factor of 12 Å 2. Each enzyme subunit is composed primarily of eight antiparallel β strands that form a flattened cylinder, plus three external loops. The β barrel is asymmetrical and can be viewed as having two distinct sides; β strands 5 to 8 are shorter with fewer hydrogen bonds, less regular side-chain alternation, and greater twist than strands 1 to 4. The main-chain hydrogen bonds primarily link β strand residues; side-chain to main-chain hydrogen bonds are extensively involved in the formation of tight turns, which form a major structural element of the three loops. The largest loop includes both a disulfide region and a Zn-liganding region, each of which resembles one of the other two loops in overall structure. The second largest loop includes a short section of α helix. The smallest loop forms a Greek key connection across one end of the β barrel. The single disulfide bond, which forms a left-handed spiral, covalently joins the largest loop to the beginning of β strand 8. Symmetrically related β bulge pairs fold the two large loops back against the external surface of the β barrel to surround the active channel. The active site Cu(II) and Zn(II) lie 6.3 Å apart at the bottom of this long channel; the Zn is buried, while the Cu is solvent-accessible. The side-chain of His61 forms a bridge between the Cu and Zn and is coplanar with them within the current accuracy of the data. The Cu ligands ND1 of His44 and NE2 of His46, −61 and −118 show an uneven tetrahedral distortion from a square plane. The Cu has a fifth axial coordination position exposed to solvent. Zn ligands ND1 of His61, −69 and −78 and OD1 of Asp81 show tetrahedral geometry with a strong distortion toward a trigonal pyramid having the buried Asp81 at the apex. Both the side-chains and mainchains of the metal-liganding residues are stabilized in their orientation by a complex network of hydrogen bonds.