Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study.
(17/174)
A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity. (+info)
Enhancement by T-type Ca2+ currents of odor sensitivity in olfactory receptor cells.
(18/174)
Mechanisms underlying action potential initiation in olfactory receptor cells (ORCs) during odor stimulation were investigated using conventional and dynamic patch-clamp recording techniques. Under current-clamp conditions, action potentials generated by a least effective odor-induced depolarization were almost completely blocked by 0.1 mm Ni(2+), a T-type Ca(2+) channel blocker, but not by 0.1 mm Cd(2+), a high voltage-activated Ca(2+) channel blocker. Under voltage-clamp conditions, depolarizing voltage steps induced a fast transient inward current, which consisted of Na(+) (I(Na)) and T-type Ca(2+) (I(Ca,T)) currents. The amplitude of I(Ca,T) was approximately one-fourth of that of I(Na) (0.23 +/- 0.03, mean +/- SEM). Because both I(Na) and I(Ca,T) are known to show rapid inactivation, we examined how much I(Na) and I(Ca,T) are activated during the gradually depolarizing initial phase of receptor potentials. The ratio of I(Ca,T)/I(Na) during a ramp depolarization at the slope of 0.5 mV/msec was 0.56 +/- 0.03. Using the dynamic patch-clamp recording technique, we also recorded I(Ca,T) and I(Na) during the generation of odor-induced action potentials. This ratio of I(Ca,T)/I(Na) was 0.54 +/- 0.04. These ratios were more than twice as large as that (0.23) obtained from the experiment using voltage steps, suggesting that I(Ca,T) carries significant amount of current to generate the action potentials. We conclude that I(Ca,T) contributes to enhance odor sensitivity by lowering the threshold of spike generation in ORCs. (+info)
Lipase-catalyzed synthesis of isoamyl butyrate. A kinetic study.
(19/174)
Kinetics of lipase-catalyzed esterification of butyric acid and isoamyl alcohol have been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. No evidence of any significant diffusional limitations was detected that could affect the kinetics. The values of the apparent kinetic parameters were computed as: V(max)=11.72 micromol/min/mg; K(M, Acid)=0.00303 M; K(M, Alcohol)=0.00306 M; K(i, Acid)=1.05 M; and K(i, Alcohol)=6.55 M. This study indicates a competitive enzyme inhibition by butyric acid during lipase-catalyzed esterification reaction. Butyric acid, being a short-chain polar acid, concentrates in the microaqueous layer and causes a pH drop in the enzyme microenvironment leading to enzyme inactivation. Butyric acid binds to acyl-enzyme complex unproductively to yield a dead-end intermediate that can no longer give rise to an ester. High concentration of butyric acid gave rise to inactivation of the biocatalyst in addition to dead-end inhibition. (+info)
Odor exposure causes central adaptation and morphological changes in selected olfactory glomeruli in Drosophila.
(20/174)
In an attempt to correlate behavioral and neuronal changes, we examined the structural and functional effects of odor exposure in Drosophila. Young adult flies were exposed to a high concentration of the selected odor, usually benzaldehyde or isoamyl acetate, for 4 d and subsequently tested for their olfactory response to a variety of odorants and concentrations. The behavioral response showed specific adaptation to the exposed odor. By contrast, olfactory transduction, as measured in electroantennograms, remained normal. In vivo volume measurements were performed on olfactory glomeruli, the anatomical and functional units involved in odor processing. Pre-exposed flies exhibited volume reduction of certain glomeruli, in an odor-selective manner. Of a sample of eight glomeruli measured, dorsal medial (DM) 2 and ventral (V) were affected by benzaldehyde exposure, whereas DM6 was affected by isoamyl acetate. Estimation of the number of synapses indicates that volume reduction involves synapse loss that can reach 30% in the V glomerulus of flies adapted to benzaldehyde. Additional features of odorant-induced adaptation, including concentration dependence and perdurance, also show correlation, because both effects are elicited by high odor concentrations and are long-lasting (>1 week). Finally, the dunce mutant fails to develop behavioral adaptation as well as morphological changes in the olfactory glomeruli after exposure. These neural changes thus appear to require the cAMP signaling pathway. (+info)
Rational design of enantioselective enzymes requires considerations of entropy.
(21/174)
Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta(R-S)DeltaH(++)) and entropy (Delta(R-S)DeltaS(++)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta(R-S)DeltaS(++) was in all cases equally significant as Delta(R-S)DeltaH(++) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++). Although the changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta(R-S)DeltaH(++) than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta(R-S)DeltaS(++). (+info)
Dual action of n-alcohols on neuronal nicotinic acetylcholine receptors.
(22/174)
Alcohol is known to modulate the activity of a variety of neuroreceptors and ion channels. Recently, neuronal nicotinic acetylcholine receptors (nnAChRs) have become a specific focus of study because not only are they potently modulated by alcohol but also they regulate the release of various transmitters, including gamma-aminobutyric acid (GABA) and dopamine, which play an important role in the behavioral effects of ethanol. Whereas the potency of normal alcohols (n-alcohols) to potentiate GABA(A) receptors and to inhibit N-methyl-D-aspartate receptors increases with carbon chain length, we have found that n-alcohols, depending on the carbon chain length, exert a dual action, potentiation and inhibition, on nnAChRs in primary cultured rat cortical neurons. The mechanism of dual action of n-alcohols on nnAChRs was further analyzed using human embryonic kidney cells expressing the alpha 4 beta 2 subunits. Shorter chain alcohols from methanol to n-propanol potentiated acetylcholine (ACh)-induced currents, whereas longer chain alcohols from n-pentanol to n-dodecanol inhibited the currents. n-Butanol either potentiated or inhibited the currents depending on the concentrations of ACh and butanol. The parameters for both potentiation (log EC(200)) and inhibition (log IC(50)) were linearly related to carbon number, albeit with different slopes. The slope for potentiation was -0.299, indicating a change in free energy change (Delta Delta G) of 405 cal/mol/methylene group, whereas the slope for inhibition was -0.584, indicating a Delta Delta G of 792 cal/mol. These results suggest that potentiating and inhibitory actions are exerted through two different binding sites. Ethanol decreased the potency of n-octanol to inhibit ACh currents, possibly resulting from an allosteric mechanism. (+info)
A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols.
(23/174)
Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth. We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step. This inhibition is strain specific and is not explained by previously described translational control pathways. Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition. Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP. This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation. (+info)
Critical time-window for NO-cGMP-dependent long-term memory formation after one-trial appetitive conditioning.
(24/174)
The nitric oxide (NO)-cGMP signaling pathway is implicated in an increasing number of experimental models of plasticity. Here, in a behavioral analysis using one-trial appetitive associative conditioning, we show that there is an obligatory requirement for this pathway in the formation of long-term memory (LTM). Moreover, we demonstrate that this requirement lasts for a critical period of approximately 5 hr after training. Specifically, we trained intact specimens of the snail Lymnaea stagnalis in a single conditioning trial using a conditioned stimulus, amyl-acetate, paired with a salient unconditioned stimulus, sucrose, for feeding. Long-term associative memory induced by a single associative trial was demonstrated at 24 hr and shown to last at least 14 d after training. Tests for LTM and its dependence on NO were performed routinely 24 hr after training. The critical period when NO was needed for memory formation was established by transiently depleting it from the animals at a series of time points after training by the injection of the NO-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO). By blocking the activity of NO synthase and soluble guanylyl cyclase enzymes after training, we provided further evidence that LTM formation depends on an intact NO-cGMP pathway. An electrophysiological correlate of LTM was also blocked by PTIO, showing that the dependence of LTM on NO is amenable to analysis at the cellular level in vitro. This represents the first demonstration that associative memory formation after single-trial appetitive classical conditioning is dependent on an intact NO-cGMP signaling pathway. (+info)