Amino Acid Substitution at the Dimeric Interface of Human Manganese Superoxide Dismutase

Hearn, Amy S.; Фан, Ли; Lepock, James R.; Luba, James; Greenleaf, William B.; Cabelli, Diane E.; Tainer, John A.; Nick, Harry S.; Silverman, David N.

doi:10.1074/jbc.m311310200

Cited by 33 publications

(33 citation statements)

References 28 publications

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“…This has been the usual observation for many other mutants of human MnSOD studied by pulse radiolysis (35, 36). We report here the new observation that for the mutants Y34A, Y34N, and Y34H there was an intermediate apparent in the catalysis.…”

Section: Resultssupporting

confidence: 57%

Contribution of Human Manganese Superoxide Dismutase Tyrosine 34 to Structure and Catalysis

et al. 2009

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Superoxide dismutase (SOD) enzymes are critical in controlling levels of reactive oxygen species (ROS) that are linked to aging, cancer and neurodegenerative disease. Superoxide (O 2 •− ) produced during respiration is removed by the product of the SOD2 gene, the homotetrameric manganese superoxide dismutase (MnSOD). Here, we examine the structural and catalytic roles of the highly conserved active-site residue Tyr34, based upon structure-function studies of MnSOD enzymes with mutations at this site. Substitution of Tyr34 with five different amino acids retained the active site protein structure and assembly, but causes a substantial decrease in the catalytic rate constant for the reduction of superoxide. The rate constant for formation of product inhibition complex also decreases but to a much lesser extent, resulting in a net increase in the product inhibition form of the mutant enzymes. Comparisons of crystal structures and catalytic rates also suggest that one mutation, Y34V, interrupts the hydrogen-bonded network, which is associated with a rapid dissociation of the productinhibited complex. Notably, with three of the Tyr34 mutants we also observe an intermediate in catalysis, which has not been reported previously. Thus, these mutants establish a means to trap a catalytic intermediate that promises to help elucidate the mechanism of catalysis.The superoxide dismutases are found in prokaryotes, archaea and eukaryotes, where they catalyze the disproportionation of the superoxide radical anion O 2•− in cellular processes detoxifying reactive oxygen species (1). The mechanism by which all superoxide dismutases carry out the catalytic removal of superoxide involves an oxidation-reduction cycle, as shown in the reactions below.In humans three SOD enzymes exist, SOD1 is a cytoplasmic Cu,ZnSOD, SOD2 is a mitochondrial MnSOD and SOD3 is an extracellular Cu,ZnSOD. These SOD enzymes are Corresponding authors: David Silverman, Phone: 352-392-3556; Fax: 352-392-9696; e-mail: silvrmn@ufl.edu Jeff Perry, Phone: 858-784-2284; Fax 858-784-2277; email jjperry@scripps.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 21. critical in controlling levels of reactive oxygen species (ROS) that are linked to aging, cancer and neurodegenerative disease (2). Structure-activity relationships for this class of enzymes need to be understood and distinguished, as their disease phenotypes likely occur through different mechanisms. SOD1 is a Cu,Zn dimeric enzyme with a β-barrel fold (3) with an active site channel containing a site for superoxide substrate and hydrogen peroxide product binding (4,5) adjacent to the activity important Arg143 side chain (6). The structure of human Cu,ZnSOD resembles that of bacterial Cu,ZnSOD except the dimer contact is on the opposite surface (7,8). For the human Cu,ZnSOD, dimer and framework-destabilizing mutations in SOD1 give rise to amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease (9-11), a progressive neurological dis...

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Section: Resultssupporting

confidence: 57%

Contribution of Human Manganese Superoxide Dismutase Tyrosine 34 to Structure and Catalysis

et al. 2009

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“…Results defining how these network side chains control the activity of hMnSOD have undergone extensive analysis, using both structure-based mutagenesis [222–226] and chemical modification approaches [227–229]. These studies revealed that any modification of residues in the hydrogen bond network affects both the activity and the stability of the enzyme, even though these alterations cause only minimal structural changes to the active site [222–225,227,230]. Mutation of hydrogen bond network residues Tyr34, His30 or Tyr166 results in a 10- to 40-fold decrease in k(cat), likely due to less efficient proton transfer to the product peroxide [225,231].…”

Section: Manganese and Iron Superoxide Dismutasesmentioning

confidence: 99%

“…These studies revealed that any modification of residues in the hydrogen bond network affects both the activity and the stability of the enzyme, even though these alterations cause only minimal structural changes to the active site [222–225,227,230]. Mutation of hydrogen bond network residues Tyr34, His30 or Tyr166 results in a 10- to 40-fold decrease in k(cat), likely due to less efficient proton transfer to the product peroxide [225,231]. Interestingly, substitution of these network amino acids may permit water molecules to replace the endogenous side chain if space allows and perform the necessary interactions to maintain the hydrogen bond network, though at these lower rates [224,227,230].…”

Section: Manganese and Iron Superoxide Dismutasesmentioning

confidence: 99%

“…One study aiming to develop such a therapeutic hMnSOD used a directed evolution approach, revealing that two mutations, C140S and N73S, result in a five-fold increase in activity of the Q143A mutant that maintains limited product inhibition [242]. Certain mutations of the hydrogen bond network partners Tyr34, His30 and Tyr166 may also be useful for generating therapeutic SOD, as they are observed to increase the dissociation of the product-inhibited complex [225,231]. Furthermore, studies on one of these mutants with reduced product inhibition, H30N, have revealed that this mutant has both anti-proliferative effects in vitro and anti-tumor effects in vivo , when it is overexpressed [243].…”

Section: Manganese and Iron Superoxide Dismutasesmentioning

confidence: 99%

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The structural biochemistry of the superoxide dismutases

Perry

Shin

Getzoff

et al. 2010

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

451

332

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The discovery of superoxide dismutases (SODs), which convert superoxide radicals to molecular oxygen and hydrogen peroxide, has been termed the most important discovery of modern biology never to win a Nobel Prize. Here, we review the reasons this discovery has been underappreciated, as well as discuss the robust results supporting its premier biological importance and utility for current research. We highlight our understanding of SOD function gained through structural biology analyses, which reveal important hydrogen-bonding schemes and metal-binding motifs. These structural features create remarkable enzymes that promote catalysis at faster than diffusion-limited rates by using electrostatic guidance. These architectures additionally alter the redox potential of the active site metal center to a range suitable for the superoxide disproportionation reaction and protect against inhibition of catalysis by molecules such as phosphate. SOD structures may also control their enzymatic activity through product inhibition; manipulation of these product inhibition levels has the potential to generate therapeutic forms of SOD. Markedly, structural destabilization of the SOD architecture can lead to disease, as mutations in Cu,ZnSOD may result in familial amyotrophic lateral sclerosis, a relatively common, rapidly progressing and fatal neurodegenerative disorder. We describe our current understanding of how these Cu,ZnSOD mutations may lead to aggregation/fibril formation, as a detailed understanding of these mechanisms provides new avenues for the development of therapeutics against this so far untreatable neurodegenerative pathology.

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“…In this case, CuZn-SOD activity outside of the matrix would be required to prevent an overwhelming inward flow of superoxide. In addition, it is known that Mn-SOD is subject to reversible product inhibition by H 2 O 2 , rendering it less active at high superoxide concentrations (51). This inhibition is quite severe in the human enzyme, less so in bacterial ones (52), and is apparently an evolutionarily conserved trait because a Mn-SOD mutant engineered to have reduced product inhibition (and thus be a more efficient dismutase) was strongly growth inhibitory when expressed in cultured cells (53).…”

Section: Figmentioning

confidence: 99%

Superoxide Inhibits 4Fe-4S Cluster Enzymes Involved in Amino Acid Biosynthesis

Wallace

Liou²,

Martins

et al. 2004

Journal of Biological Chemistry

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Among the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy; in the current work we show an additional leaky auxotrophy for leucine. The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively. We present evidence that direct aerobic inactivation of these enzymes in sod1⌬ yeast results in the auxotrophies. Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p. Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix. The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1⌬ mutants. We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial "free" (EPRdetectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities. Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.The antioxidant enzyme copper/zinc-superoxide dismutase 1 plays an integral role in the protection of many organisms from the oxidative aerobic environment.CuZn-SOD is localized to the cytosol, nucleus, and the intermembrane space (IMS) of the mitochondria, suggesting that it exerts its protective effect in multiple compartments (1). Another SOD that contains manganese (Mn-SOD or Sod2p) is located in the matrix of mitochondria. Saccharomyces cerevisiae lacking CuZn-SOD (sod1⌬) have distinct and well established aerobic phenotypes including diminished growth, auxotrophies for lysine and either methionine or cysteine, decreased ability to grow on nonfermentable carbon sources (2, 3), increased "free" (EPR-detectable) iron (4), hypersensitivity to millimolar concentrations of zinc (5), and exquisite sensitivity to redox-cycling drugs such as the herbicide paraquat (6, 7). All of these phenotypes (except zinc sensitivity) are also observed in mutants lacking the copper chaperone for CuZn-SOD 2 (lys7⌬ or ccs1⌬) and thus contain a form of the Sod1p polypeptide that is inactive because of a lack of copper in the active site (5, 8). Mutants lacking Sod2p have a less dramatic phenotype; they are sensitive to redox cycling drugs and grow poorly on nonfermentable carbon sources but show no aerobic auxotrophies (2).Many cellular components are susceptible to oxidative damage, including proteins, lipids, and DNA (9, 10). However, targets of superoxide-specific damage are much more limited. A particular type of protein prosthetic group, solvent-exposed 4Fe-4S clusters occurring in nonelectron transfer proteins, have been shown to be specifically damaged by superoxid...

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Amino Acid Substitution at the Dimeric Interface of Human Manganese Superoxide Dismutase

Cited by 33 publications

References 28 publications

Contribution of Human Manganese Superoxide Dismutase Tyrosine 34 to Structure and Catalysis

Contribution of Human Manganese Superoxide Dismutase Tyrosine 34 to Structure and Catalysis

The structural biochemistry of the superoxide dismutases

Superoxide Inhibits 4Fe-4S Cluster Enzymes Involved in Amino Acid Biosynthesis

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