Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

Abstract

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Å, 100K; 0.80 Å, 15K; 1.75 Å, 293K), neutron Laue data (2.2 Å, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.enzymatic mechanism ͉ helium cooling ͉ subatomic resolution crystallography ͉ x-ray plus neutrons joint refinement

The mobile proton hypothesis in fragmentation of protonated peptides: A perspective

“…The generalized concept of mobile protons in chemistry has a long history, in that the Grotthuss mechanism [27] for the anomalously high conductivity of pure water was published in 1806 and is still the subject of active research [28,29]. In enzyme catalysis, the importance of the intramolecular mobility of protons has earned a different name, "proton translocation" [30]. "Mobile protons" are now an essential feature in understanding the physico-chemical properties of certain organic crystals [31], and similarly in the development of SiO 2 thin films as a potential rival for chips based on Si [32].…”

Room-temperature ultrahigh-resolution time-of-flight neutron and X-ray diffraction studies of H/D-exchanged crambin

“…More importantly, the neutron scattering length of deuterium (+6.671 fm) is comparable to those of oxygen (+5.803 fm), carbon (+6.646 fm) and nitrogen (+9.36 fm), while hydrogen has a negative scattering length (À3.7406 fm). This property has been utilized to accurately locate H atoms of critical interest in enzyme mechanisms (Kossiakoff & Spencer, 1981;Bennett et al, 2006;Blakeley et al, 2008;Kovalevsky et al, 2008 and also to obtain information on the dynamics of proteins through their backbone amide-exchange characteristics (Kossiakoff, 1982). Furthermore, neutron crystallography has been successful in resolving water species such as D 2 O, OD À and D 3 O + , orienting hydrogen-bond donors and acceptors, and for orienting metal-coordinated solvent molecules (Blum et al, 2009;Chen et al, 2010;Fisher et al, 2010;Kovalevsky et al, 2011).…”

X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction

“…The unique information available from neutron crystallography has provided important insights into the mechanisms of enzymes Bennett et al, 2006;Blakeley et al, 2008;Blum et al, 2009;Coates et al, 2001Coates et al, , 2008Katz et al, 2006;Kossiakoff & Spencer, 1981;Kovalevsky et al, 2008;Tamada et al, 2009;Yagi et al, 2009;Tomanicek et al, 2010). Until recently, elucidating neutron structures has required large amounts of sample in order to grow large crystals (typically >1 mm 3 ) and long datacollection periods.…”

“…Complete perdeuteration of proteins, which was first demonstrated for staphylococcal nuclease (Gamble et al, 1994) and myoglobin (Shu et al, 2000) and has subsequently been applied to aldose reductase (Hazemann et al, 2005;Blakeley et al, 2008) and type III antifreeze protein (Petit-Haertlein et al, 2009), has allowed the collection of neutron diffraction data from crystals as small as $0.1 mm 3 . Encouraged by these results, several investigators are exploring perdeuteration as a means of making neutron crystallographic analyses feasible for proteins that can only be grown to small crystal volumes or to improve the resolution of neutron data collected from deuterated crystals of hydrogenous protein (Artero et al, 2005;Budayova-Spano et al, 2006;Liu et al, 2007;Meilleur et al, 2004Meilleur et al, , 2005Tuominen et al, 2004).…”

“…Until recently, elucidating neutron structures has required large amounts of sample in order to grow large crystals (typically >1 mm 3 ) and long datacollection periods. Recent developments in instrumentation and data-collection techniques at both reactor and spallation neutron sources (Blakeley et al, 2008;Langan et al, 2008) have significantly reduced the sample-size requirements and data-acquisition times for neutron data sets (Blum et al, 2007). Concurrently, recent improvements in structure-refinement software and strategies, such as the use of X-ray and neutron crystallographic data in maximum-likelihood refinement in nCNS and PHENIX, have helped to address the limitations of the low data-to-parameter ratios and low data resolutions that are often associated with neutron crystallographic data (Adams et al, 2009).…”