Rachel V. Dunn

Enzyme Activity below the Dynamical Transition at 220 K

Enzyme activity requires the activation of anharmonic motions, such as jumps between potential energy wells. However, in general, the forms and time scales of the functionally important anharmonic dynamics coupled to motion along the reaction coordinate remain to be determined. In particular, the question arises whether the temperature-dependent dynamical transition from harmonic to anharmonic motion in proteins, which has been observed experimentally and using molecular dynamics simulation, involves the activation of motions required for enzyme function. Here we present parallel measurements of the activity and dynamics of a cryosolution of glutamate dehydrogenase as a function of temperature. The dynamical atomic fluctuations faster than approximately 100 ps were determined using neutron scattering. The results show that the enzyme remains active below the dynamical transition observed at approximately 220 K, i.e., at temperatures where no anharmonic motion is detected. Furthermore, the activity shows no significant deviation from Arrhenius behavior down to 190 K. The results indicate that the observed transition in the enzymes dynamics is decoupled from the rate-limiting step along the reaction coordinate.

Enzyme dynamics and activity: time-scale dependence of dynamical transitions in glutamate dehydrogenase solution.

We have examined the temperature dependence of motions in a cryosolution of the enzyme glutamate dehydrogenase (GDH) and compared these with activity. Dynamic neutron scattering was performed with two instruments of different energy resolution, permitting the separate determination of the average dynamical mean square displacements on the sub-approximately 100 ps and sub-approximately 5 ns time scales. The results demonstrate a marked dependence on the time scale of the temperature profile of the mean square displacement. The lowest temperature at which anharmonic motion is observed is heavily dependent on the time window of the instrument used to observe the dynamics. Several dynamical transitions (inflexions of the mean squared displacement) are observed in the slower dynamics. Comparison with the temperature profile of the activity of the enzyme in the same solvent reveals dynamical transitions that have no effect on GDH function.

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme–substrate complex. However, recent steady‐state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady‐state and stopped‐flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme–substrate complex attributed to a deprotonation event required for optimal catalysis with a pKa of 7.4–8.4. In stopped‐flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell‐shaped pH profile for the rate of flavin reduction over the substrate binding constant (kred/Ks, reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pKa of the free amine is above 9.3 for all substrates, and is greatly perturbed (ΔpKa∼ 2) on binding to the enzyme active site. This perturbation of the substrate amine pKa on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme–substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.

Enzyme activity down to −100°C

Abstract The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to −97°C and −100°C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below −100°C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.

Tyrosyl Radical Formation and Propagation in Flavin Dependent Monoamine Oxidases

Understanding the mechanism of biological amine oxidation by flavoproteins remains a controversial area of research. This is particularly true for the mammalian monoamine oxidases (MAO), which are major pharmaceutical targets for the development of antidepressants and neuroprotective agents. Despite intensive research efforts, the detailed reaction mechanism, which will have major implications for drug discovery with this class of enzyme, has yet to be discovered. Mechanistic debates have centred on the potential involvement of radical species in the activity of these enzymes. We now provide strong evidence that supports a role for radical catalysis in flavoprotein monoamine oxidases. The mammalian monoamine oxidases (EC 1.4.3.4) are localised to the outer mitochondrial membrane. In these enzymes, the flavin cofactor, FAD, is covalently linked through the 8a-methyl group to an active site cysteine residue. A number of mechanisms for MAO-catalysed amine oxidation have been proposed over the years, and several reviews are available. There are currently three main mechanistic proposals for MAO catalysis. These comprise: 1) the concerted polar nucleophilic mechanism; 2) the direct hydride transfer mechanism; and 3) the single electron transfer mechanism. Recent support for the concerted polar nucleophilic mechanism has come from kinetic and structural studies on tyrosine mutants of MAO B, and also from computational studies. 9] However, analysis of the nitrogen isotope effects conducted on a related amine oxidase, Nmethyltryptophan oxidase, supported either a direct hydride transfer mechanism or, possibly, a discrete electron transfer mechanism. The single electron transfer (SET) mechanism initially proposed by Silverman and colleagues involves single electron transfer from the substrate nitrogen lone pair to yield the substrate radical and flavin semiquinone. The SET mechanism is consistent with electronic effects observed in quantitative structure activity relationships studies with a series of substituted benzylamines, but the identity of the one-electron oxidant required for the formation of the aminyl radical cation was a major concern. Recent spectroscopic evidence for the presence of a stable tyrosyl radical in partially reduced human MAO A provided support for the SET mechanism and led to the proposal of a modified SET mechanism (Scheme 1). After the initial single-

Activity and Dynamics of an Enzyme, Pig Liver Esterase, in Near-Anhydrous Conditions

Water is widely assumed to be essential for life, although the exact molecular basis of this requirement is unclear. Water facilitates protein motions, and although enzyme activity has been demonstrated at low hydrations in organic solvents, such nonaqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an enzyme, pig liver esterase, to catalyze alcoholysis as an anhydrous powder, in a reaction system of defined water content and where the substrates and products are gaseous. At hydrations of 3 (±2) molecules of water per molecule of enzyme, activity is several orders-of-magnitude greater than nonenzymatic catalysis. Neutron spectroscopy indicates that the fast (≤nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are suppressed. This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.

Cryosolvents useful for protein and enzyme studies below −100°C

Abstract For the study of protein structure, dynamics, and function, at very low temperatures it is desirable to use cryosolvents that resist phase separation and crystallisation. We have examined these properties in a variety of cryosolvents. Using visual and X-ray diffraction criteria, methanol:ethanediol (70%:10%), methanol:glycerol (70%:10%), acetone:methoxyethanol:ethanediol (35%:35%:10%), dimethylformamide:ethanediol (70%:10%), dimethylformamide (80%), methoxyethanol (80%), and methoxyethanol:ethanediol (70%:10%) were all found to be free of phase-changes down to at least −160°C. The least viscous of these, methanol:ethanediol (70%:10%), was miscible down to −125°C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology.

Proton tunnelling and promoting vibrations during the oxidation of ascorbate by ferricyanide

A combination of the temperature- and pressure-dependencies of the kinetic isotope effect on the proton coupled electron transfer during ascorbate oxidation by ferricyanide suggests that this reference reaction may exploit vibrationally assisted quantum tunnelling of the transferred proton.