Energetics and dynamics of the proton shuttle of carbonic anhydrase II

Abstract

Human carbonic anhydrase II catalyzes the reversible reaction of carbon dioxide and water to form bicarbonate and a proton. His64-mediated proton shuttling between the active site and the bulk solvent is rate limiting. Here we investigate the protonation behavior of His64 as well as its structural and dynamic features in a pH dependent way. We derive two pKa values for His64, 6.25 and 7.60, that we were able to assign to its inward and outward conformation. Furthermore, we show that His64 exists in both conformations equally, independent of pH. Both conformations display an equal distribution of their two neutral tautomeric states. The life time of each conformation is short and both states display high flexibility within their orientation. Therefore, His64 is never static, but rather poised to change conformation. These findings support an energetic, dynamic and solution ensemble-based framework for the high enzymatic activity of human carbonic anhydrase II.

pH-Dependent Structure and Dynamics of the Catalytic Domains of Human Carbonic Anhydrase II and IX

“…The mechanism outlined above is presented in Figure b with additional details added from a large body of simulation studies at neutral pH. ,− Two major dynamical reorganization processes have been found to be crucial in initiating the actual proton transfer across the active site: (i) rapid fluctuations of the coordination number of zinc ion between 4 and 5 and (ii) fast rotation of His-64 side chain between its in and out orientations. The fast kinetics associated with His-64 side chain rotation has recently been confirmed by nuclear magnetic resonance (NMR) studies . Subsequently, reorganization of water molecules at the active site is found to facilitate transfer of the proton fluctuating between its Eigen and Zundel forms ,,, These dynamical fluctuations have been shown to be necessary to reproduce the experimental value of k cat (∼0.8–1.25 × 10 6 μs –1 ) .…”

“…Earlier experimental observations implied a pK a of 7.1 for His-64 in HCA II. 69 In a more recent study, 43 NMR spectroscopy within the pH range 5−10 reported for the first time two distinct pK a values of 6.25 and 7.60 associated with its in and out conformations, respectively. Our estimates, despite a nonequilibrium sampling reported at pH 5 and 6, corroborate well to the conformationdependent values of pK a as obtained from the NMR titration of His-64 side chain in HCA II.…”

“…The fast kinetics associated with His-64 side chain rotation has recently been confirmed by nuclear magnetic resonance (NMR) studies. 43 Subsequently, reorganization of water molecules at the active site is found to facilitate transfer of the proton fluctuating between its Eigen and Zundel forms 36,38,40,42 These dynamical fluctuations have been shown to be necessary to reproduce the experimental value of k cat (∼0.8−1.25 × 10 6 μs −1 ). 8 Although His-64 has been detected only in its out side chain conformation in HCA IX-c, 15 our earlier studies indicated that similar to the side chain in HCA II, the His-64 side chain can undergo fast reorientation at neutral pH.…”

Proton Transfer Kinetics in Histidine Side Chains Determined by pH-Dependent Multi-Nuclear NMR Relaxation

“…These features make histidine the chemically most versatile amino acid, and it is often engaged in catalysis. 4,5 In fact, histidine has many different functions: it can act as a nucleophile and as an acid/base catalyst, 6,7 e.g., in catalytic triads, 8,9 as a proton shuttle, 10,11 as a hydrogen bond donor and acceptor, 12,13 and it can coordinate metal ions. 14 Hydrogen bonding involving histidine is of particular interest in the context of protein structure.…”