Kinetic and Structural Characterization of Spinach Carbonic Anhydrase

Rowlett, Roger S.; Chance, Mark R.; Wirt, Michael D.; Sidelinger, Dean E.; Royal, Jill R.; Woodroffe, Melanie; Wang, Ying Fuh A; Saha, Rajib; Lam, Michael G.

doi:10.1021/bi00251a003

Cited by 84 publications

(76 citation statements)

References 57 publications

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“…EXAFS results suggest that the active site zinc of ␤-class Cab is coordinated by two cysteine residues and two oxygen/nitrogen ligands, with the possibility that one of the oxygen/nitrogen ligands derives from histidine (Table 2, fit 4). Our results are nearly identical to EXAFS results previously reported for the S. oleracea enzyme (11,46). Cys-32, His-87, and Cys-90 of Cab are completely conserved in all known ␤-class carbonic anhydrase sequences (Fig.…”

Section: Discussionsupporting

confidence: 91%

“…The kinetic properties reported for the P. sativum and Spinacia oleracea (spinach) ␤-class carbonic anhydrases are also consistent with this mechanism (29,30,46). Thus, the kinetic analyses of enzymes from all three classes suggest convergent evolution of the catalytic mechanism (1,36,39).…”

supporting

confidence: 54%

“…Nearby ionizable groups may influence these pH profiles to produce multiple pK a values, or the pK a values are lower than 6.0. The steady-state parameter k cat of the ␤-class S. oleracea carbonic anhydrase was found to be pH dependent, with an apparent pK a of approximately 8.5 in the absence of sulfate (46). Similar to Cab, the pH profiles for both k cat and k cat /K m in the direction of CO 2 hydration for the ␤-class P. sativum carbonic anhydrase are pH dependent, although the profiles were not fitted to theoretical titration curves (29).…”

Section: Discussionmentioning

confidence: 97%

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Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

et al. 2000

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The ␤-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) ␤-class carbonic anhydrase indicate that the secondary structure of the ␤-class enzymes is predominantly ␣-helical, unlike that of the ␣-or ␥-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k cat and k cat /K m for CO 2 hydration are pH dependent. The steady-state parameter k cat is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k cat is 2.1-fold higher in H 2 O than in D 2 O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k cat /K m is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic ␤-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic ␤-class enzymes and firmly establish that the ␣-, ␤-, and ␥-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.The thermophilic archaeon Methanobacterium thermoautotrophicum obtains energy for growth by the reduction of CO 2 to CH 4 and is also an obligate chemolithoautotroph; thus, this organism has a high demand for CO 2 . Carbonic anhydrase, a zinc-containing enzyme catalyzing the reversible hydration of carbon dioxide (equation 1)is expected to play an important role in the growth of M. thermoautotrophicum and may have several functions, including transporting HCO 3 Ϫ into the cell and providing CO 2 or HCO 3 Ϫ to enzymes that utilize these substrates. Based on sequence comparisons, carbonic anhydrases belong to three genetically distinct classes (␣, ␤, and ␥) which appear to have independent origins (24). The most extensively studied enzymes are those from the ␣-class, which is composed primarily of mammalian carbonic anhydrases, but also includes enzymes from the green alga Chlamydomonas reinhardtii (19,20) and the prokaryote Neisseria gonorrhoeae (13). The ␤-class enzymes are abundant in C 3 and C 4 monocotyledenous and dicotyledenous plants and green unicellular algae (24, 43), where they are essential for photosynthetic CO 2 fixation (6). The most recently identified class of carbonic anhydrase, the ␥-class (24), is represented by the prototype Cam from the archaeon Methanosarcina thermophila (2). Even though sequences encoding putative ␥-class carbonic anhydrases have been found in prokaryotes from both the Bacteria and Archaea domains (2...

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Section: Discussionsupporting

confidence: 91%

supporting

confidence: 54%

Section: Discussionmentioning

confidence: 97%

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Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

et al. 2000

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“…Very recently, the crystal structures of a b-CAs from Porphyridium purpureum (Mitsuhashi et al 2000) and Pisum sativum (Kimber & Pai, 2000) were resolved. The pea CA is an octamer in which dimers form tetramers that form octamers.Active sites are at the subunit interfaces and as suggested by EXAFS, mutagenesis and elemental analysis (Rowlett et al 1994;Bracey et al 1994), the zinc is bound by two cysteines and one histidine. In the algal CA an aspartate rather than water occupies the fourth co-ordination position (Mitsuhashi et al 2000).…”

Section: B -Carbonic Anhydrasesmentioning

confidence: 99%

Carbonic anhydrases in plants and algae

Moroney¹,

Bartlett²,

Samuelsson³

2001

Plant Cell Environ

108

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Carbonic anhydrases catalyse the reversible hydration of CO 2 , increasing the interconversion between CO 2 and HCO 3 -+ H + in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated a-, b-and g-CA. Animals have only the a-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C 3 dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.

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“…Although the polypeptide assembly forming the active site of the ␥-CA is different from those of the ␣-CAs, the geometry around the zinc of ␥-CA is quite similar to those of ␣-CAs. (9,10), indicating that the higher plant ␤-CAs are catalytically as efficient as human CA II. On the other hand, extended x-ray absorption fine structure suggests that the coordination sphere of zinc in ␤-CAs of higher plants should have one or more sulfur ligands (10,11) in contrast to those in ␣-and ␥-CAs.…”

mentioning

confidence: 99%

X-ray Structure of β-Carbonic Anhydrase from the Red Alga,Porphyridium purpureum, Reveals a Novel Catalytic Site for CO2 Hydration

Mitsuhashi¹,

Mizushima²,

Yamashita³

et al. 2000

Journal of Biological Chemistry

165

168

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The carbonic anhydrases (CAs) fall into three evolutionarily distinct families designated ␣-, ␤-, and ␥-CAs based on their primary structure. ␤-CAs are present in higher plants, algae, and prokaryotes, and are involved in inorganic carbon utilization. Here, we describe the novel x-ray structure of ␤-CA from the red alga, Porphyridium purpureum, at 2.2-Å resolution using intrinsic zinc multiwavelength anomalous diffraction phasing. The CA monomer is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an ␣/␤ domain and three projecting ␣-helices. The motif is obviously distinct from that of either ␣-or ␥-CAs. This homodimeric CA appears like a tetramer with a pseudo 222 symmetry. The active site zinc is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the ␤-CAs. No water molecule is found in a zinc-liganding radius, indicating that the zinc-hydroxide mechanism in ␣-CAs, and possibly in ␥-CAs, is not directly applicable to the case in ␤-CAs. Zinc coordination environments of the CAs provide an interesting example of the convergent evolution of distinct catalytic sites required for the same CO 2 hydration reaction.Carbonic anhydrase (CA, 1 EC 4.2.1.1) is a zinc-containing enzyme which catalyzes the reversible hydration of CO 2 . CA is ubiquitously distributed in nature and is involved in fundamental biological processes such as photosynthesis, respiration, pH homeostasis, and ion transport (1, 2). Based on sequence homologies, CAs are classified into three evolutionarily distinct groups, designated ␣-, ␤-, and ␥-CAs (3). ␣-CAs are found in mammals, algae, and prokaryotes, ␤-CAs in higher plants, algae, and prokaryotes, and ␥-CA has been identified in the archaeon, Methanosarcina thermophila, although gene homologies have been recently found in higher plants and prokaryotes (4).The x-ray structures of ␣-CAs from several mammals (5, 6) have revealed a common overall structure dominated by ␤-sheets. The catalytically active zinc is liganded by three histidine residues with a hydroxide or a water molecule as a fourth ligand, giving a tetrahedral coordination geometry. For the CO 2 hydration reaction, the zinc-bound hydroxide initiates a nucleophilic attack on the substrate CO 2 to form zinc-bound HCO 3 Ϫ which is then displaced by a water molecule (7). The regeneration of zinc-bound hydroxide requires an intramolecular proton shuttle from the zinc-bound water to the bulk solvent for each cycle of catalysis. The proton transfer is ratelimiting in the catalysis (8).The x-ray structure of ␥-CA from M. thermophila has been reported (4). This is a trimeric CA with a peculiar left-handed ␤-helical folding motif. The active site zinc is located at subunit interfaces and coordinated by two histidines from one subunit and one histidine from a neighboring one. In addition, at an electron dense region which occupies the fourth coordination site, a putative water molecule is recognized. Although the polypeptide assembly forming the active site of the ...

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Kinetic and Structural Characterization of Spinach Carbonic Anhydrase

Cited by 84 publications

References 57 publications

Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

Carbonic anhydrases in plants and algae

X-ray Structure of β-Carbonic Anhydrase from the Red Alga,Porphyridium purpureum, Reveals a Novel Catalytic Site for CO2 Hydration

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