Kinetic Analysis of Multiple Proton Shuttles in the Active Site of Human Carbonic Anhydrase

Abstract

We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His 7 in HCA III. Using an 18 O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His 7 and His 64 within the active site cavity, shows the conformation of the side chain of His 64 moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His 7 . This suggests a cause of decreased proton transfer in catalysis.The carbonic anhydrases in the ␣ class include the mammalian isozymes and are all zinc-containing monomeric enzymes, generally with molecular masses near 30 kDa. These isozymes of carbonic anhydrase catalyze the dehydration/hydration of HCO 3 Ϫ /CO 2 by a two-stage or ping-pong mechanism in which the first step (Equation 1) is the reaction of bicarbonate with the enzyme containing zinc-bound water resulting in the formation of CO 2 and leaving zinc-bound hydroxide at the active site (1, 2).The second stage is the regeneration of the zinc-bound water through proton transfer (1, 2). BH ϩ represents a proton donor that is a residue of the enzyme itself or buffer in solution. In the carbonic anhydrases, the maximal velocities of catalysis and the rates of exchange of 18 O between CO 2 and water are limited by the intramolecular proton transfers indicated in Equation 2. This was initially determined by observation of a solvent deuterium isotope effect of 3.8 on k cat for hydration catalyzed by human carbonic anhydrase (HCA) 1 II, among the most efficient isozymes in the ␣ class of carbonic anhydrases (3). The solvent hydrogen isotope effect was 2.4 for 18 O exchange catalyzed by HCA III, the least efficient of the ␣ class (4). Subsequent results including pH profiles, buffer activation, and computer simulations for these isozymes support the rate-limiting nature of proton transfer in these isozymes (4 -7).For HCA II, the catalytic turnover is near 10 6 s Ϫ1 . For this isozyme the predominant shuttle residue has been identified as His 64 (3, 5), the side chain of which extends into the active site cavity without apparent interactions with other residues of the protein (8, 9). The imidazole ring of His 64 is about 7.5 Å from the zinc when this side chain is in the "in" conformation pointing toward the metal (8). This is too far for direct proton transfe...

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

“…After noticing the flexible conformation of the His64 side chain (Krebs et al, 1991; at different pH, the proton transfer pathway was proposed and confirmed analyzing numerous mutants of CA II by McKenna and Christianson groups Scolnick and Christianson, 1996;Tu et al, 2002;Bhatt et al, 2005Zheng et al, 2008;Domsic et al, 2010;Mikulski et al, 2011aMikulski et al, , 2011bMikulski et al, , 2013. Taylor et al (1970), that the on-rate k a depends on pH in a similar fashion as the thermodynamic K D .…”

“…Technique -Inhibition FTSA, ITC CA I (Morkūnaitė et al, 2015) 8.1 (Coleman, 1967) 8.4 ± 0.2 8.1 −47.9 −48.2 −41.0 −38.5 6.9 9.7 6.9 (Innocenti et al, 2009) 7.1 (Engstrand et al, 1995) 7.3 (Whitney and Brandt, 1976;Khalifah, 1977;Khalifah et al, 1977) CA II (Morkūnaitė et al, 2015) 6.9 (Duda et al, 2001;Mikulski et al, 2011a) 7.1 6.9 −40.5 −41.1 −26.0 −23.5 14.5 17.6 7.1 (Innocenti et al, 2009) 6.8 (Tu et al, 2002) CA III ∼5 (Tu et al, 2002) 6.6 6.5 −37.7 −38.6 ND ND ND ND…”

High Resolution Crystal Structures of Human Cytosolic Thiolase (CT): A Comparison of the Active Sites of Human CT, Bacterial Thiolase, and Bacterial KAS I

“…Currently, the functional importance of such an array of water molecules for efficient catalysis is unclear; however, a linear array of water molecules can function as a proton wire. [51][52] In the thiolase active site, proton transfer to the negatively charged thioester oxyanion, which is predicted to be formed transiently both during the acetyl-transfer step as well as the Claisen condensation step, might stabilise the transition state. Comparison of the active sites of CT, bacterial thiolase, and KAS I The Claisen condensation reaction ( Figure 1) is a common feature of the catalytic cycles of thiolases and KAS enzymes.…”

Substrate-dependent Interference of Carbonic Anhydrases with the Glutamine Transporter SNAT3-Induced Conductance

“…CAIII, which lacks the H + shuttling histidine at position 64, has very little enzymatic activity in vitro, as has also been determined for the mutant CAII-H64A [14][15][16]. The current traces in Fig.…”