Franco Marmocchi

Crystal structure of yeast Cu,Zn superoxide dismutase: Crystallographic refinement at 2.5 Å resolution☆

The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 A resolution. The enzyme crystallizes in the P2(1)2(1)2 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 A resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 A from the ideal bond lengths, and an average B-factor value of 29.9 A2. A dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded beta-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 beta-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c beta-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 beta-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu2+ ligands show an uneven distortion from a square plane, while Zn2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 A away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers.(ABSTRACT TRUNCATED AT 400 WORDS)

A study of the pH dependence of the activity of porcine Cu,Zn superoxide dismutase

Abstract The enzyme activity of porcine Cu,Zn superoxide dismutase, which has an unusually high isoelectric point, decreases almost linearly with increasing pH between pH 7.5 and 12.0, while EPR and NMR parameters of the copper are titrated only above pH 9.5. Elimination of lysine charges by succinylation abolishes the pH dependence of activity between pH 7.5 and 9.5 and produces identical pH-activity curves for both bovine and porcine enzymes. The pH dependence of activity of the succinylated proteins overlaps that of the spectroscopic parameters of the copper in both enzymes. These results indicate that lysines play a critical role in the rate determining step of the mechanism of the Cu,Zn superoxide dismutases.

Inhibition of adenosine deaminase from several sources by deaza derivatives of adenosine and EHNA.

Deaza analogues of adenosine and EHNA were tested as inhibitors of the enzyme adenosine deaminase (ADA) obtained from several sources including human erythrocytes, calf intestine, Saccaromices cerevisiae, Escherichia coli and Takadiastase. Ki values of the inhibitors suggest differences among the enzymes both at purine and erythro-nonyl binding site. Among the ribofuranosyl derivatives, 1-deazaadenosine is the best inhibitor, its Ki ranging between 3.5 x 10(-7) and 4 x 10(-5) M for ADA from erythrocytes and Takadiastase respectively. Only ADA from erythrocytes and calf intestine bind EHNA and some of deazaEHNA analogues; 3-deazaEHNA behaves very similarly to EHNA both in affinity and slow binding mechanism, whereas 1-deazaEHNA, though less potent, is a good inhibitor.

A comparative study of bovine, porcine and yeast superoxide dismutases

SummaryThe Cu,Zn superoxide dismutases from bovine and porcine erythrocytes and from yeast have been investigated with the aim to identify structural differences in relation to possible functional variability in this highly homologous class of protein. The isoelectric points of the bovine, porcine and yeast proteins were found to be 4.8, 5.8 and 4.5 respectively. According to these values the net protein charge, as evaluated by gel electrophoresis, varied more significantly for the porcine protein than for the other two proteins tested. The catalytic constants were found to be higher at pH = 7.6 than at pH = 10.0 for all the three enzymes. This relative increase was much more pronounced in the case of the porcine enzyme. The KM value at pH = 10.0 was also significantly higher for the porcine enzyme. Since the spectroscopic properties of the active sites were identical for the three proteins, these results point to modulation effects by positively charged amino acid residues on the superoxide dismutase activity of these proteins, in a way that the resultant net charge of the protein seems to be as important as specific residues.

DIFFERENTIAL EFFECTS OF UREA ON YEAST AND BOVINE COPPER, ZINC SUPEROXIDE DISMUTASES, IN RELATION TO THE EXTENT OF ANALOGY OF PRIMARY STRUCTURE

Abstract Incubation in 8M urea (pH 7.4) inactivated yeast Cu, Zn superoxide dismutase with biphasic first order kinetics (k for the decrease from 100% to 16% activity = 6.5 × 10 −3 min −1 ; k for the decrease from 16% to 0.1% activity = 2.5 × 10 −3 min −1 ). The inactivation was fully reversible on dilution with or dialysis against urea-free buffer. No inactivation was shown to occur in similar experiments with the bovine Cu, Zn enzyme. EPR spectra recorded immediately after addition of 8M urea showed a more axial line shape and a higher A ″ of the copper signal typical of the native enzyme. In the case of the yeast enzyme, this change was more pronounced and further incubation led to a new type of copper signal, typical of the inactivated enzyme. All EPR changes were reversible. Comparative analysis of the amino acid sequence of the two enzymes showed substantial identity of the protein regions contributing the ligands to the metals and the disulfide bridge. Differential destabilization of active sites by urea should be due to replacements in other protein segments, such as the three C-terminal and some N-terminal residues.

Dissociation of Cu-Zn superoxide dismutase into monomers by urea: Evidence from gel filtration and molecular hybridization

Copper-zinc superoxide dismutases are dimeric enzymes with mol. wt -33 000, which contain 1 functional copper and 1 auxiliary zinc, located close to each other on identical subunits [I] . In spite of spectroscopic and structural equivalence of the copper binding sites, the copper ion on one subunit influences equilibrium and kinetic properties of the copper [2]. Intersubunit interaction appear to be also relevant to the catalytic mechanism [3]. Thus, obtaining active monomers of copper-zinc superoxide dismutase is vital to the understanding of the enzyme mechanism, especially considering that monomeric superoxide dismutases have never been discovered. Sodium dodecyl sulfate (SDS) has been used to attempt dissociation of these enzymes and has been found to have different effects on various superoxide dismutases. While the bovine enzyme, which is the most extensively studied, has been reported to be resistant to dissociation in SDS [4] partially active monomers (< 50% native enzyme activity) have been obtained by SDS treatment of one isoenzyme from wheat germ [S] . Evidence for dissociation in 8 M urea of a fish superoxide dismutase has been presented on the basis of a gel filtration experiment 161. In this report we show that dissociation into monomers of copper-zinc superoxide dismutases by 8 M urea seems to be a general property of this class of enzymes and indicates a peculiar sensitivity of their quaternary structure to this denaturing agent.

Functional residues at the active site of bovine brain adenosine deaminase

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first‐order kinetics with a second‐order rate constant of 8.9 10‐3 (±1.8 10‐3) M‐1 min‐1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern‐Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground‐ and transitionstate analogs to the enzyme.

Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase

The Cu,Zn superoxide dismutase from yeast was crystallized in the orthorhombic space group P21212 with unit cell dimension a = 105.1 A,b = 142.2 A, c = 62.1 A. The crystals grow in 25 mM citrate, 10 mM phosphate buffer pH 6.5, and 6% (W/V) polyethylene glycol, with a Vm of 3,4 A3/dalton, for two dimers/asymmetric unit. The crystals were unstable in the mother liquor, but were stabilized by transfer to a 35% polyethylene glycol solution. This crystalline form diffracts at high resolution and is suitable for determination of the atomic structure. The three dimensional structure of the yeast enzyme could be model-built by computer graphics techniques using the bovine enzyme atomic coordinates as template. The proposed model requires removal of some salt bridges and non equivalence of the metal-binding sites in the subunits, in line with reported functional properties of the yeast enzyme.

Enzyme activity of superoxide dismutase protomers

Superoxide dismutase from eucaryotic cells are dimeric enzymes of 33 000 mol. wt. containing 1 cupric copper and 1 zinc per protomer [l] . The copper site is the active site, while the zinc has a structural role [2,3] The enzymes purified from mammalian cells are considerably resistant to denaturating agents such as 4% sodium dodecyl sulfate (SDS) and 10 M urea [3] . In particular SDS is not able to dissociate the mammalian enzymes into protomers of 16 000 mol. wt. in the absence of 2-mercaptoethanol, although it has been shown that no covalent linkages exist between the two subunits [4]. Recently it has been reported [4] that wheat germ contains two cupro-zinc isozymes, which differ from each other in so far that one of them dissociates into protomers in SDS even in the absence of 2-mercaptoethanol. As the activity of the dimer is unaffected by SDS while treatment with SDS and 2-mercaptoethanol produces inactive protomers [4] it seemed worthwhile to test the activity of the protomers obtained by the action of SDS alone. Recent studies on the mechanism of superoxide dismutase [5] suggested that only half the active sites of the bovine enzyme function in catalysis, even though the two copper centers are indistinguishable in terms of spectral features [ 51 and anion reactivity [6] This may indicate a flip-flop type of mechanism [7], in which the reaction of one copper with a superoxide ion renders the other unreactive. In the light of these considerations, we now report on the enzyme activity of wheat germ superoxide dismutase as affected by SDS and subsequent removal of the denaturating agent.

Inhibition studies on membrane adenosine deaminase from human placenta

The ecto form of adenosine deaminase isolated from human placental membrane was tested towards its sensitivity against adenosine deaminase inhibitors, such as aza and deaza analogues of adenosine and erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA). Ki values of the inhibitors observed were similar to these obtained for the small form of adenosine deaminase purified from human erythrocytes, indicating that the presence of the binding protein on placental adenosine deaminase does not produce alteration in the binding of these inhibitors on the enzyme active site. The inhibition rate of 2′‐deoxycoformycin, one of the most potent ADA inhibitors is affected by the presence of the binding protein on human placental adenosine deaminase, that probably modulates the rearrangment of the active site produced by the binding with this tight‐binding inhibitor.