A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus. A kinetic model for the enzyme action

Abstract

The enzymic reaction mechanism of a manganese-containing superoxide dismutase from Bacillus stearothermophilus was studied by using pulse radiolysis. During catalysis (pH 8.9; 25 degrees C), changes occurring in the kinetics of substrate disappearance and in the visible absorption of the enzyme at 480 nm established that the simple two-step mechanism found for copper- and iron-containing superoxide dismutases is not involved. At a low ratio (less than 15) of substrate concentration to enzyme concentration the decay of O2--is close to exponetial, whereas at much higher ratios (greater than 100) the observed decay is predominantly zero-order. The simplest interpretation of the results invokes a rapid one-electron oxidation-reduction cycle ('the fast cycle') and, concurrently, a slower reaction giving a form of the enzyme that is essentially unreactive towards O2-- but which undergoes a first-order decay to yield fully active native enzyme ('the slow cycle'). The fast cycle involves the native enzyme EA and a form of the enzyme EB which can be obtained also by treating the form EA with H2O2. Computer calculations made with such a simple model predict behaviour in excellent agreement with the observed results.

Characterization of the Product-inhibited Complex in Catalysis by Human Manganese Superoxide Dismutase

“…Unlike its analog Fe-SOD, which is inactivated in the presence of H 2 O 2 through the Fenton-type chemistry in which H 2 O 2 reacts with iron (14), Mn-SOD is not a redox catalyst for Fenton chemistry. The result that Mn-SOD is not irreversibly inactivated with H 2 O 2 is consistent with earlier reports of McAdam et al (15) and more recent studies of Yamakura et al (16). DISCUSSION The rapid mixing of H 2 O 2 with human Mn(III)SOD and subsequent scanning stopped-flow spectrophotometry has resulted in the appearance of a visible absorption spectrum (Fig.…”

“…The larger of the firstorder rate constants, 7 s Ϫ1 , is close to k Ϫ5 ϭ 10 s Ϫ1 determined by Bull et al (3) with wild type. The models for catalysis of both McAdam et al (5) and Bull et al (3) relate the magnitude of inhibition and the zero-order rate constant of the inhibited phase to this decay rate designated k Ϫ5 of Equation 2. However, it is important to point out that many of the rate constants of Equations 1 and 2 besides k Ϫ5 could also possibly contribute to the greater product inhibition of Y34F Mn-SOD observed during the catalyzed dismutation of O 2 .…”

Synthesis, X-Ray Crystallographic Characterization, and Electronic Structure Studies of a Di-Azide Iron(III) Complex: Implications for the Azide Adducts of Iron(III) Superoxide Dismutase

“…1,2 Mn-and FeSODs, like all other SODs, disproportionate the superoxide radical anion into dioxygen and hydrogen peroxide in a two-step ping-pong-type mechanism (eqs 1a and 1b, where M = Fe or Mn) during which the metal ion cycles between the 2+ and 3+ oxidation states. [3][4][5] While structurally unrelated to the Cu/Zn-and NiSODs, Mn-and FeSODs possess nearly identical protein folds and highly homologous active sites. 6 In the resting states of both proteins, the metal ions are in five-coordinate, distorted trigonal bipyramidal ligand environments with a histidine (His) and a solvent molecule in the axial positions and two His residues and an aspartate (Asp) in the equatorial plane (Figure 1, left).…”

Potent Anti-tumor Effects of an Active Site Mutant of Human Manganese-Superoxide Dismutase

“…are converted to O 2 and H 2 O 2 by either the uninhibited or inhibited (Mn(X)-SOD) pathways (15). The product-inhibited state of Mn-SOD has been conserved from bacteria to man with the emergence of the inhibited form appearing 30-fold more rapidly for the human enzyme (11).…”