Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer

Abstract

Human carbonic anhydrase II (HCA II) uses a Zn-bound OH − /H 2 O mechanism to catalyze the reversible hydration of CO 2 . This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pK a compared with free tyrosine. To further probe the pK a of this residue, NMR spectroscopic measurements of [ 13 C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pK a of Tyr7 and how pH and Tyr proximity to Zn affect hydrogenbonding interactions.proton transfer | neutron crystallography | perturbed pKa | solution NMR C arbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the interconversion of CO 2 and HCO 3 − . In humans, CAs are involved in a great variety of physiological processes, from respiration, ureagenesis, gluconeogenesis, and cerebrospinal fluid production to general acid/base homeostasis (1). The most abundant and well-characterized human CA isoform is human carbonic anhydrase II (HCA II), which displays a bellshaped pH dependence on catalytic activity, with a maximal k cat of 10 6 per second at pH 7.2. In the hydration direction, the first step of catalysis is a Zn-OH − -mediated nucleophilic attack on CO 2 to form HCO 3 − and one H + , leaving one water molecule bound to the zinc. The second, rate-limiting step occurs via proton transfer (PT) that removes one H + from the metal-bound water to regenerate the catalytic Zn-OH − (2). This PT step is thought to adopt classic Grotthuss-type hopping and it is mediated by a well-ordered network of hydrogen-bonded (H-bonded) water molecules that spans the ∼8-Å distance between the Znwater and proton-shuttling residue, His64 (Figs. 1-3). In previous neutron structures, the Zn-bound solvent has been observed to be an H 2 O (or exchanged D 2 O) and this may imply that H 2 O is the favored species over OH − in the absence of substrate (3). In chemical crystallography extended X-ray absorption fine structure can be used to determine Zn:Zn-solvent distances as a way to discriminate between H 2 O or OH − . For Zn-OH − the distance should be ∼1.80 Å and for Zn-H 2 O it should be closer to 2.0 Å (4). Despite the availability of ultra-high-resolution X-ray crystal structures of holo HCA II (no substrate present) the range of Zn:Zn-solvent distances observed has led to inconclusive results. Also, within the coordinate error it is unfeasible to determine the Zn-bound solvent species using distance with any certainty (5, 6).His64 is thought to be one component in the delivery of excess H + to the bulk solvent, resetting the a...

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations

“…Before calculations, hydrogen atoms, which were not visible in the crystallographic structures, were added to the models and their positions were energy minimised using the Discover module of InsightII package. It is worth of note that in all the protonated complexes, in agreement with what observed in the neutronic structure of hCA II crystallised at pH 7.5 (PDB code 4Q49) 72 , the hydrogen bound to the Thr200OG1 atom was oriented towards Pro201O atom, in a direction opposite to the position of the ligand (Figure 4). Consequently, the Thr200OG1 atom can act only as a hydrogen bond acceptor when interacting with the ligand.…”

Neutron protein crystallography: A complementary tool for locating hydrogens in proteins

“…Neutron crystallography is a powerful complementary approach to NMR spectroscopy and X-ray crystallography. Neutron crystallography and NMR spectroscopic measurements following the titration of tyrosine residues were elegantly combined in a study of carbonic anhydrase by Michalczyk et al [26]. NMR revealed the perturbed pK a of Tyr 7, and the neutron crystal structures provided a structural context.…”

“…Despite a number of significant technical challenges that we will discuss in this review, the ability to experimentally locate deuterium makes neutron diffraction a method of choice for visualizing the positions of hydrogen in enzymes, whether used as a standalone technique or in complementary with Nuclear Magnetic Resonance (NMR) spectroscopy or high resolution X-ray crystallography. Neutron protein crystallography has, for example, been used to locate hydrogen atoms and determine the protonation states at the active site of HIV-1 protease (HIV Pr) [16,17], beta-lactamase [18][19][20], dihydrofolate reductase (DHFR) [21,22], carbonic anhydrase [23][24][25][26], xylose isomerase [27][28][29][30][31][32][33]. These studies have been essential in identifying active site residues with perturbed pK a 's and contribute to the growing body of knowledge on pK a modulation at the active site of enzymes.…”

Neutron Crystallography for the Study of Hydrogen Bonds in Macromolecules

“…This indicates that H bonds cannot be identified only based on geometric criteria and that some additional structural or chemical knowledge is needed to fully understand all the H bonds in a protein. In recent years, bifurcated H bonds have also been directly observed in neutron protein crystal structures between Tyr and solvent, and Lys and Thr [ 9 , 10 ].…”

“…Further neutron structures were solved at pH 7.8 and 6.0 to further probe the solvent network and changes in protonation of Tyr7 ( Figure 3 b,c) [ 9 , 24 ]. The pH 7.8 neutron structure showed a rearrangement of the solvent network, with all the waters now connected by H bonds, and neutral His64 poised to accept a proton.…”