Structures of calcineurin and its complexes with immunophilins-immunosuppressants

Calcineurin (CN) is a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase and is involved in many physiological processes such as T-cell activation and cardiac hypertrophy. The crystal structures of CN and its complexes with FKBP12-FK506 and cyclophilin-cyclosporin showed that the two structurally unrelated immunophilins-immunosuppressants bind to a common composite surface made up of the residues from both catalytic subunit and regulatory subunit of CN. The recognition of the immunophilins and immunosuppressive drugs is achieved by common but few distinct CN residues. However, the binding pattern of FKBP12-FK506 such as hydrogen bonding is significantly different from that of CyPA-CsA. This common but distinct recognition may indicate capacity of the composition surface for binding of other inhibitory proteins. The recognition site and the active site are adjacent and form an "L" shaped cleft. This implies that the immunophilin recognition site may also serve as a recognition site to define the narrow substrate specificity of calcineurin.     

Interfacial versus homogeneous enzymatic cleavage of mandelonitrile by hydroxynitrile lyase in a biphasic system

The question of an interfacial versus a homogeneous reaction is carefully addressed for the enzymatic biphasic cleavage of mandelonitrile to benzaldehyde by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) (Hickel et al. [1999] Biotechnol Bioeng 36:425-436). Experimental evidence, including 1) the reaction ceases when the interface is populated by previously adsorbed denatured pa-Hnl, 2) the reaction continues even after washout of the bulk enzyme from the aqueous phase, 3) highly nonpolar organic solvents initially promote fast reaction kinetics that relatively quickly decay to zero product production, and 4) the reaction rate is nonlinear in the bulk enzyme concentration, provide robust grounds for an interfacial reaction. We also model enzymatic mandelonitrile cleavage assuming a homogeneous aqueous-phase reaction. The homogeneous reaction scheme does not simultaneously account for the experimental observations of a linear dependence of the reaction rate on organic/water interfacial area, no dependence on the aqueous-phase volume, and a nonlinear dependence on pa-Hnl aqueous concentration. Further, simple calculations demonstrate that the homogeneous reaction rate is at least three orders of magnitude slower than those observed by Hickel et al. (1999). We again conclude that enzyme adsorbed at the organic solvent/water interface primarily catalyzes the biphasic mandelonitrile cleavage reaction.     

Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study

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.     

Proton transfer dynamics of GART: the pH-dependent catalytic mechanism examined by electrostatic calculations

The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pK(a) of approximately 8. Experimental studies of pH-rate profiles of wild-type and site-directed mutants of GART have led to the proposal that His108, Asp144, and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pK(a) of 9.7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)s of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pK(a) calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, catalysis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histidine, and a low pK(a) for GAR(NH(2)) was calculated, indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART deduced from the experimental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static time-averaged crystallographic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH(2)) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pK(a) of GAR(NH(2)) modeled from the experimental data using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.     

Asymmetric catalytic ene-cyclization approach to 2-fluoro-19-nor-1,25-dihydroxyvitamin D(3) A-ring analog with significant transactivation activity

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)2D3) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for the treatment of cancer and psoriasis. However, little information has been available on the binding conformation of the 1alpha,25(OH)2D3 molecule and its analogs with the vitamin D receptor (VDR). Therefore, we synthesized 2alpha-fluorinated A-ring analogs of 19-nor-1alpha,25(OH)2D3 in order to investigate the VDR-binding conformation of the A-rings on the basis of the (19)F NMR analysis. The 2alpha-fluoro-19-nor-1alpha,25-dihydroxyvitamin D3 A-ring analog thus synthesized via a asymmetric catalytic carbonyl-ene cyclization, shows significant activity in transactivation.     

A kinetic model for enzyme interfacial activity and stability: pa-hydroxynitrile lyase at the diisopropyl ether/water interface

A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.     

Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films

Protein-chitosan (CS) films were made by casting a solution of proteins and CS on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) incorporated in CS films gave a pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks at about -0.33V vs saturated calomel electrode in pH 7 buffers, respectively, while catalase (Ct) in CS films showed a peak pair at about -0.46V which was not stable. All these peaks are located at the potentials characteristic of heme Fe(III)/Fe(II) redox couples of the proteins. The electrochemical parameters such as formal potentials (E degrees (')) and apparent heterogeneous electron-transfer rate constants (k(s)) were estimated by square-wave voltammetry with nonlinear regression analysis. Chitosan films contained considerable water and formed hydrogel in aqueous solution. Positions of the Soret absorbance band suggest that Mb and Hb in CS films keep their secondary structure similar to the native states in the medium pH range, while HRP and Ct retain their native conformation at least in the dry CS films. Scanning electron microscopy of the films demonstrated that interaction between the proteins and CS would make the morphology of dry protein-CS films very different from the CS films alone. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all four proteins in CS films.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.     

Montmorillonite Catalysis of 30–50 Mer Oligonucleotides: Laboratory Demonstration of Potential Steps in the Origin of the RNA World

Elongation of the primer ³²pdA(pdA)₈pA proceeds by thereaction of the 5-phosphorimidazolides of adenosine and uridine in the presence of montmorillonite clay. Daily addition of the activated nucleotides for up to 14 days results in theformation of 40–50 mers using the 5-phosphorimidazolide of adenosine (ImpA) and 25–30 mers using the 5-phosphorimidazolide of uridine (ImpU). The limitation on thelengths of the chains formed is not due to the inhibitors formedsince the same chain lengths were formed using 2–3 times the amount of montmorillonite catalyst. The shorter oligomers formedby the addition of U monomers is not due to its greater rate ofdecomposition since it was found that both the A and the U adducts decompose at about the same rates. Alkaline phosphatase hydrolysis studies revealed that some of the oligomers are cappedat the 5-end to form, with ImpA,Ap³²pdA(pdA)₈pA(pA)_n. The extent of capping depends on the reaction time and the purine or pyrimidine base inthe activated mononucleotide. Hydrolysis with ribonuclease T₂ followed by alkaline phosphatase determined the sites ofthe 3, 5- and 2, 5-phosphodiester bonding to the primer. The potential significance of the mineral catalyzed formation of 50 mer oligonucleotides to the origin of life basedon RNA (the RNA world scenario) is discussed.     

Monitoring the kinetics and thermodynamics of interfacial enzymatic catalysis by differential scanning calorimetry

Using phase transition profile as an indicator of thermodynamic property and phase transition heat as the second indicator of the percentage of substrates unhydrolyzed, differential scanning calorimetry has been used to observe in detail the kinetics and thermodynamics of phospholipase A(2)-catalyzed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine large unilamellar vesicle (LUV) hydrolysis. Phase transition profiles show that the original LUV almost completely changes into a novel aggregate at the end of the latency, followed by an abrupt activation of the reaction. The phase transition profiles are asymmetric between the heating and cooling curves, indicating a thermodynamic mesostatic property of the system. The reaction in activated phase follows a single first-order kinetics and all of the substrates in vesicles can be hydrolyzed. All these evidences indicate that the products and substrates can freely exchange between the outer and the inner layers of the vesicles and the membrane of the vesicle in the activated phase is permeable. This permeability favors the exchange of the substrates and products, thus, resulting in the activation of the fast reaction.