Intramolecular proton shuttle supports not only catalytic but also noncatalytic function of carbonic anhydrase II

Abstract

Carbonic anhydrases (CAs) catalyze the reversible hydration of CO 2 to HCO 3 − and H + . The rate-limiting step in this reaction is the shuttle of protons between the catalytic center of the enzyme and the bulk solution. In carbonic anhydrase II (CAII), the fastest and most wide-spread isoform, this H + shuttle is facilitated by the side chain of His64, whereas CA isoforms such as carbonic anhydrase III (CAIII), which lack such a shuttle, have only low catalytic activity in vitro. By using heterologous protein expression in Xenopus oocytes, we tested the role of this intramolecular H + shuttle on CA activity in an intact cell. The data revealed that CAIII, shown in vitro to have ∼1,000-fold reduced activity as compared with CAII, displays significant catalytic activity in the intact cell. Furthermore, we tested the hypothesis that the H + shuttle in CAII itself can facilitate transport activity of the monocarboxylate transporters 1 and 4 (MCT1/4) independent of catalytic activity. Our results show that His64 is essential for the enhancement of lactate transport via MCT1/4, because a mutation of this residue to alanine (CAII-H64A) abolishes the CAII-induced increase in MCT1/4 activity. However, injection of 4-methylimidazole, which acts as an exogenous H + donor/acceptor, can restore the ability of CAII-H64A to enhance transport activity of MCT1/4. These findings support the hypothesis that the H + shuttle in CAII not only facilitates CAII catalytic activity but also can enhance activity of acid-/base-transporting proteins such as MCT1/4 in a direct, noncatalytic manner, possibly by acting as an “H + -collecting antenna.”

Analysis of the Binding Moiety Mediating the Interaction between Monocarboxylate Transporters and Carbonic Anhydrase II

“…In contrast to the transport metabolons described so far, this interaction is independent of CAII catalytic activity but is mediated by an intramolecular H ϩ shuttle within the enzyme (47,48). This led to the conclusion that CAII, directly bound to the transporter, can act as a H ϩ -collecting/distributing antenna, which shuttles protons between transporter and surrounding protonatable residues to support proton/lactate cotransport (48). By introduction of single-site mutations in the C terminus of MCT1 and subsequent expression of these mutants in CAII-injected Xenopus oocytes, we could identify the two glutamic acidic residues Glu 489 and Glu…”

“…39 -41. Using heterologous protein expression in Xenopus oocytes, we have recently reported that CAII enhances transport activity of MCT1 and MCT4, while leaving transport activity of MCT2 unaffected (42)(43)(44)(45)(46). In contrast to the transport metabolons described so far, this interaction is independent of CAII catalytic activity but is mediated by an intramolecular H ϩ shuttle within the enzyme (47,48). This led to the conclusion that CAII, directly bound to the transporter, can act as a H ϩ -collecting/distributing antenna, which shuttles protons between transporter and surrounding protonatable residues to support proton/lactate cotransport (48).…”

GPI-anchored carbonic anhydrase IV displays both intra- and extracellular activity in cRNA-injected oocytes and in mouse neurons

“…Thus, our results suggest that CAs, known to be active extracellularly, also display robust intracellular activity. Therefore, the intracellular CA activity, which is crucial for cellular H + dynamics and various acid/base transport processes across the cell membrane (20,26), is not only due to known, soluble cytosolic CAs, but is also attributable to intracellular activity of membrane-associated CAs. This discovery makes it important to take into account the interplay between intracellular and extracellular CA activities to understand their role in physiological processes and to design drugs specific for extracellular CA isoforms.…”

“…In addition, evidence about the catalytic activity of CA in oocytes, either expressed or injected as protein, can be obtained physiologically by measuring intracellular H + in intact oocytes, and by mass spectrometry of lysed oocytes (20,21). By measuring H + with ion-selective microelectrodes in the cytosol and at the outer membrane surface, intra-and extracellular CA activity can be identified and discriminated by the rate and amplitude, respectively, of H + changes.…”

Synthesis and characterization of phenolic Mannich bases and effects of these compounds on human carbonic anhydrase isozymes I and II

“…Since CA inhibitors have been shown to reduce IOP exclusively by lowering the aqueous humour flow and these compounds have been used for the treatment of glaucoma for years 11,12 . The rate-determining step for the CO 2 hydration reaction catalyzed by CAs is the proton transfer reaction from the water bound to the Zn(II) ion to the reaction medium, with generation of the zinc hydroxide species of the enzyme [13][14][15][16][17][18][19][20] . Several anions behave as inhibitors for the CO 2 hydration reaction 21,22 and coordinate directly to Zn(II) ion.…”