Chemical Rescue in Catalysis by Human Carbonic Anhydrases II and III

An, Haiqian; Tu, Chingkuang; Duda, David M.; Montañez-Clemente, Ileana; Math, Kristen; Laipis, Philip J.; McKenna, Robert; Silverman, David N.

doi:10.1021/bi0120695

Cited by 63 publications

(121 citation statements)

References 35 publications

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“…Perhaps such weak binding would indicate little change in pK a of the amine upon binding. This was the case with activating derivatives of imidazole and pyridine during catalysis by carbonic anhydrase (29).…”

Section: Resultsmentioning

confidence: 98%

“…General Acid Catalysis and Fe-SOD-A role for exogenous proton donors and acceptors in catalysis is well studied in enzymatic reactions that consume or generate protons, such as the hydration/dehydration of CO 2 /HCO 3 Ϫ catalyzed by carbonic anhydrase (29,30) and the activation of a site-specific mutant of aspartate aminotransferase (31). However, there is an interesting difference between activation of Fe-SOD and these examples.…”

Section: Discussionmentioning

confidence: 99%

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Activation of the Proton Transfer Pathway in Catalysis by Iron Superoxide Dismutase

Greenleaf

Silverman

2002

Journal of Biological Chemistry

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Catalysis by Escherichia coli and Porphyromonas gingivalis iron superoxide dismutase was activated by addition of primary amines, as measured by pulse radiolysis and stopped-flow spectrophotometry. This activation was saturable for most amines investigated, and a free energy plot of the apparent second-order rate constant of activation was linear as a function of the pK a of the amine, indicating activation by proton transfer. Amines provide an alternate rather than the only pathway for proton transfer, and catalysis was appreciable in the absence of amines. Solvent hydrogen isotope effects were near unity for amine activation, which is consistent with rate-contributing proton transfer if the pK a of the proton acceptor on the enzyme is not in the region of the pK a values of the amines studied, from 7.8 to 10.6. The activation of catalysis by these amines was uncompetitive with respect to superoxide, interpreted as proton transfer in a ternary complex of amine with the enzyme-bound peroxide dianion.The superoxide dismutases (SODs) 1 are cyclic redox enzymes that provide protection against oxidative damage caused by the reactions of the superoxide radical anion O 2 . . SODs have been characterized with manganese, iron, copper/zinc, or nickel as the redox active metal (1-3). The catalysis of the dismutation of superoxide occurs in two stages in which the metal ion, designated M in Reactions 1 and 2, alternates between the M 3ϩ and M 2ϩ forms (2-4),Iron superoxide dismutase (Fe-SOD), found in prokaryotes, and Mn-SOD, found in mitochondria as well as in prokaryotes, are highly similar in sequence (5) and in crystal structure (6, 7). Despite this high degree of structural homology, substitution of one metal for the other generally results in an inactive enzyme (8,9). A subclass of SOD exists that is able to utilize either manganese or iron as catalytic metal ion; these are termed cambialistic SODs and are of significant interest from the perspective of metal specificity as well as catalytic mechanism. A well studied cambialistic SOD is that from Porphyromonas gingivalis, the crystal structure of which is reported for the manganese-and iron-containing forms (10). Although they all share common features of their catalytic pathways, the copper/ zinc-and nickel-containing SODs have structures unrelated to the manganese-and iron-containing SODs (2, 11).The crystal structures of the iron-and manganese-containing superoxide dismutases generally show a trigonal bipyramidal geometry about the oxidized metal with three histidines, one aspartic acid, and one solvent molecule as ligands (6, 7). Major features of the active site appear very similar in each enzyme, including the highly conserved hydrogen-bonded network connecting the metal-coordinated solvent molecule and residues Gln-69 2 (in Fe-SOD) or Gln-143 (in Mn-SOD), Tyr-34, His-30, and Tyr-166 from the adjacent subunit. Although this active site Gln emanates from a different backbone position in Fe-SOD and Mn-SOD, the side chain orientation is very similar in both. Thi...

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Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Activation of the Proton Transfer Pathway in Catalysis by Iron Superoxide Dismutase

Greenleaf

Silverman

2002

Journal of Biological Chemistry

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“…Thus, Mt-CamH was overproduced in E. coli and characterized to begin to investigate the CamH subclass and increase our overall understanding of the ␥ class and the physiological role of CAs in Methanosarcina species. Mt-CamH was overproduced in media supplemented with either ZnSO 4 or FeSO 4 and was purified to homogeneity as determined by SDS-PAGE. The N terminus of the proteins overproduced with either metal supplement was identical to the first 15 amino acid residues of the sequence deduced from the gene encoding Mt-CamH.…”

Section: Resultsmentioning

confidence: 99%

Characterization of CamH from Methanosarcina thermophila , Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

2010

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The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the ␥ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had k cat and k cat /K m values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-MtCamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective k cat (k cat eff ) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k cat /K m eff was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k cat eff of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H 2 O 2 , which supports the hypothesis that Fe 2؉ has a role in the active site. The k cat for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a "ping-pong" mechanism accepting a proton. The k cat /K m was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO 2 and HCO 3 ؊ is separate from intermolecular proton transfer.

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“…The low barrier found for the W2 case is similar to the intrinsic barrier found in the recent experimental studies of the proton transfer between H64A CA II and derivatives of imidazole and pyridine in solution. 50 Moreover, Lu and Voth 33 found in their classical MD simulation that a value of 2-3 kcal/mol should be added for the organization of the water bridge. This is one aspect of the possible contributions of the protein environment, which was not included in the current analysis.…”

Section: Concluding Discussionmentioning

confidence: 99%

Is a “Proton Wire” Concerted or Stepwise? A Model Study of Proton Transfer in Carbonic Anhydrase

2003

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The energetics of proton transfer reactions in carbonic anhydrase (CA) have been studied with an active site model. Specifically, proton transfer from a zinc-bound water molecule to a histidine residue mediated by a numbers of water molecules was investigated. With two or three bridging water molecules, the proton transfers are fully or nearly fully concerted and only one saddle point exists. With an additional water molecule that forms a ring bridge, an intermediate is formed in which one of the water molecules exists as a hydronium ion. In contrast to previous calculations in which either a low-level of theory was employed or a stepwise mechanism was assumed, the energetics obtained from the current work are approximately consistent with the experimental estimates. In all of the scenarios, the motion of more than one proton is involved in the transition state, which is in agreement with the experimental observation that the reaction rates in H 2 O/D 2 O mixture have an exponential dependence on the fraction of D 2 O in the solvent. For three (W3) or four waters (W4), the proton transfer to the "His 64" model is hardly involved in the transition state, suggesting that the orientation of the proton acceptor is less important than for only two waters (W2). Thus, the W3 and W4 results are consistent with the experimental observation that many kinetic properties of the H64A mutant of CA in well-buffered imidazole solution are similar to the wild type. The barrier height increases, and the barrier frequency (and therefore, the contribution of tunneling) decreases as the number of bridging water molecules increases. Overall, these investigations demonstrate that the proton transfer reaction in CA is sensitive to the nature and structure of the water bridge, which would be influenced by the dynamics of the water molecules and amino acids in the active site of the protein.

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Chemical Rescue in Catalysis by Human Carbonic Anhydrases II and III

Cited by 63 publications

References 35 publications

Activation of the Proton Transfer Pathway in Catalysis by Iron Superoxide Dismutase

Activation of the Proton Transfer Pathway in Catalysis by Iron Superoxide Dismutase

Characterization of CamH from Methanosarcina thermophila , Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

Is a “Proton Wire” Concerted or Stepwise? A Model Study of Proton Transfer in Carbonic Anhydrase

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