Comparative Kinetic Study Between Native and Chemically Modified Cu,Zn Superoxide Dismutases

Overview

Journal Biochem J

Specialty Biochemistry

Date 1993 Jun 1

PMID 8503879

Citations 1

Authors

E Argese

R Girotto

E F Orsega

Affiliations

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Abstract

The kinetic behaviour of native bovine erythrocyte Cu,Zn superoxide dismutase (N-SOD) and of its derivatives by reaction with polyethylene glycol, acetic and succinic anhydrides has been investigated here in detail. Their responses to changes of pH and ionic strength (I) have been used as a probe for quantitatively displaying the relevance to kinetic rate constant of superficial positive charges driving the superoxide ion (O2-) toward the enzyme's active site. Overall kinetic trends indicate that this long-range O2- electrostatic guidance is essentially due to the positive charges of the amino-acid residues Lys-120 and Lys-134 which are strategically located around the active site. The comparison between the kinetic data obtained from N-SOD and those from polyethylene-glycolated SOD (PEG-SOD) enabled us to state that in PEG-SOD an O2(-)-steering positive electrostatic force, halved in comparison with N-SOD, is still operating, and that only Lys-120 is linked in the reaction of N-SOD with PEG. Elimination of the electrostatic driving force, carried out either by deprotonation of lysine amino groups at high pH, or by their neutralization with succinic anhydride and acetic anhydride, or by ionic screening at high ionic strength, always lowered the kinetic rate constant to a value of approx. 3 x 10(8) M-1.s-1. This value is about 15 times smaller than that measured in the presence of the reactant-steering mechanism and represents the k value of the reaction limited by pure diffusion. Finally, the kinetic behaviour of acetylated SOD and succinylated SOD demonstrated the inhibitor effect of OH- at strongly alkaline pH.

Citing Articles

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Gabdoulline R, Stein M, Wade R BMC Bioinformatics. 2007; 8:373.

PMID: 17919319 PMC: 2174957. DOI: 10.1186/1471-2105-8-373.

References

McCord J, Fridovich I . Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem. 1969; 244(22):6049-55. View

Sines J, Allison S, McCammon J . Point charge distributions and electrostatic steering in enzyme/substrate encounter: Brownian dynamics of modified copper/zinc superoxide dismutases. Biochemistry. 1990; 29(40):9403-12. DOI: 10.1021/bi00492a014. View

Fridovich I, RABANI J . Pulse radiolytic investigations of superoxide catalyzed disproportionation. Mechanism for bovine superoxide dismutase. J Am Chem Soc. 1973; 95(9):2786-90. DOI: 10.1021/ja00790a007. View

Fielden E, Roberts P, Bray R, Lowe D, Mautner G, Rotilio G . Mechanism of action of superoxide dismutase from pulse radiolysis and electron paramagnetic resonance. Evidence that only half the active sites function in catalysis. Biochem J. 1974; 139(1):49-60. PMC: 1166250. DOI: 10.1042/bj1390049. View

Malinowski D, Fridovich I . Bovine erythrocyte superoxide dismutase: diazo coupling, subunit interactions, and electrophoretic variants. Biochemistry. 1979; 18(1):237-44. DOI: 10.1021/bi00568a037. View

Boden N, Holmes M, Knowles P . Properties of the cupric sites in bovine superoxide dismutase studied by nuclear-magnetic-relaxation measurements. Biochem J. 1979; 177(1):303-9. PMC: 1186369. DOI: 10.1042/bj1770303. View

Viglino P, Rigo A, Argese E, Calabrese L, Cocco D, Rotilio G . F19 relaxation as a probe of the oxidation state of Cu, Zn superoxide dismutase. Studies of the enzyme in steady-state turnover. Biochem Biophys Res Commun. 1981; 100(1):125-30. DOI: 10.1016/s0006-291x(81)80072-1. View

Bertini I, Luchinat C, Messori L . A water 17O NMR study of the pH dependent properties of superoxide dismutase. Biochem Biophys Res Commun. 1981; 101(2):577-83. DOI: 10.1016/0006-291x(81)91298-5. View

Marmocchi F, Mavelli I, Rigo A, Stevanato R, Bossa F, Rotilio G . Succinylated copper, zinc superoxide dismutase. A novel approach to the problem of active subunits. Biochemistry. 1982; 21(12):2853-6. DOI: 10.1021/bi00541a007. View

10.

Cudd A, Fridovich I . Electrostatic interactions in the reaction mechanism of bovine erythrocyte superoxide dismutase. J Biol Chem. 1982; 257(19):11443-7. View

11.

Tainer J, Getzoff E, Beem K, Richardson J, Richardson D . Determination and analysis of the 2 A-structure of copper, zinc superoxide dismutase. J Mol Biol. 1982; 160(2):181-217. DOI: 10.1016/0022-2836(82)90174-7. View

12.

Cocco D, Rossi L, Barra D, Bossa F, Rotilio G . Carbamoylation of Cu,Zn-superoxide dismutase by cyanate. Role of lysines in the enzyme action. FEBS Lett. 1982; 150(2):303-6. DOI: 10.1016/0014-5793(82)80756-4. View

13.

Cocco D, Mavelli I, Rossi L, Rotilio G . Reduced anion binding and anion inhibition in Cu, Zn superoxide dismutase chemically modified at lysines without alteration of the rhombic distortion of the copper site. Biochem Biophys Res Commun. 1983; 111(3):860-4. DOI: 10.1016/0006-291x(83)91378-5. View

14.

Getzoff E, Tainer J, Weiner P, Kollman P, Richardson J, Richardson D . Electrostatic recognition between superoxide and copper, zinc superoxide dismutase. Nature. 1983; 306(5940):287-90. DOI: 10.1038/306287a0. View

15.

Veronese F, Largajolli R, Boccu E, Benassi C, Schiavon O . Surface modification of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase. Appl Biochem Biotechnol. 1985; 11(2):141-52. DOI: 10.1007/BF02798546. View

16.

Allison S, Ganti G, McCammon J . Simulation of the diffusion-controlled reaction between superoxide and superoxide dismutase. I. Simple models. Biopolymers. 1985; 24(7):1323-36. DOI: 10.1002/bip.360240717. View

17.

Borders Jr C, Saunders J, Blech D, Fridovich I . Essentiality of the active-site arginine residue for the normal catalytic activity of Cu,Zn superoxide dismutase. Biochem J. 1985; 230(3):771-6. PMC: 1152682. DOI: 10.1042/bj2300771. View

18.

Fee J, Bull C . Steady-state kinetic studies of superoxide dismutases. Saturative behavior of the copper- and zinc-containing protein. J Biol Chem. 1986; 261(28):13000-5. View

19.

Sharp K, Fine R, Honig B . Computer simulations of the diffusion of a substrate to an active site of an enzyme. Science. 1987; 236(4807):1460-3. DOI: 10.1126/science.3589666. View

20.

Argese E, Viglino P, Rotilio G, Scarpa M, Rigo A . Electrostatic control of the rate-determining step of the copper, zinc superoxide dismutase catalytic reaction. Biochemistry. 1987; 26(11):3224-8. DOI: 10.1021/bi00385a043. View