XFEL structure of carbonic anhydrase II: a comparative study of XFEL, NMR, X-ray and neutron structures.

The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.

Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase.

Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3 -) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (k cat and k cat/K m) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure-function relationships of many other biologically important enzymes.

Elucidating the role of metal ions in carbonic anhydrase catalysis.

Why metalloenzymes often show dramatic changes in their catalytic activity when subjected to chemically similar but non-native metal substitutions is a long-standing puzzle. Here, we report on the catalytic roles of metal ions in a model metalloenzyme system, human carbonic anhydrase II (CA II). Through a comparative study on the intermediate states of the zinc-bound native CA II and non-native metal-substituted CA IIs, we demonstrate that the characteristic metal ion coordination geometries (tetrahedral for Zn2+, tetrahedral to octahedral conversion for Co2+, octahedral for Ni2+, and trigonal bipyramidal for Cu2+) directly modulate the catalytic efficacy. In addition, we reveal that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring water network in the active site. Our study provides evidence that the metal ions in metalloenzymes have a crucial impact on the catalytic mechanism beyond their primary chemical properties.

Analysis of the effect of oral midazolam and triazolam premedication before general anesthesia in patients with disabilities with difficulty in cooperation.

BACKGROUND: When performing dental treatment under general anesthesia in adult patients who have difficulty cooperating due to intellectual disabilities, anesthesia induction may be difficult as well. In particular, patients who refuse to come into the dental office or sit in the dental chair may have to be forced to do so. However, for adult patients with a large physique, physical restraint may be difficult, while oral sedatives as premedication may be helpful. Here, a retrospective analysis was performed to investigate the effect of oral sedatives. METHODS: A hospital-based medical information database was searched for patients who were prescribed oral midazolam or triazolam between January 2009 and December 2017. Pre-anesthesia evaluation, anesthesia, and anesthesia recovery records of all patients were analyzed, and information on disability type, reason for prescribing oral sedatives, prescribed medication and dose, cooperation level during anesthesia induction, anesthesia duration, length of recovery room stay, and complications was retrieved. RESULTS: A total of 97 patients were identified, of whom 50 and 47 received midazolam and triazolam, respectively. The major types of disability were intellectual disabilities, autism, Down syndrome, blindness, cerebral palsy, and epilepsy. Analyses of changes in cooperation levels after drug administration showed that anesthesia induction without physical restraint was possible in 56.0% of patients in the midazolam group and in 46.8% of patients in the triazolam group (P = 0.312). CONCLUSIONS: With administration of oral midazolam or triazolam, general anesthesia induction without any physical restraint was possible in approximately 50% of patients, with no difference between the drugs.

Carbonic anhydrase II microcrystals suitable for XFEL studies.

Recent advances in X-ray free-electron laser (XFEL) sources have permitted the study of protein dynamics. Femtosecond X-ray pulses have allowed the visualization of intermediate states in enzyme catalysis. In this study, the growth of carbonic anhydrase II microcrystals (40-80 µm in length) suitable for the collection of XFEL diffraction data at the Pohang Accelerator Laboratory is demonstrated. The crystals diffracted to 1.7 Šresolution and were indexed in space group P21, with unit-cell parameters a = 42.2, b = 41.2, c = 72.0 Å, ß = 104.2°. These preliminary results provide the necessary framework for time-resolved experiments to study carbonic anhydrase catalysis at XFEL beamlines.