A designed synthetic analogue of 4-OT is specific for a non-natural substrate

The substrate specificity of 4-oxalocrotonate tautomerase (4-OT) is characterized by electrostatic interactions between positively charged arginine (Arg) side chains on the enzyme and the dianionic substrate, 4-oxalocrotonate. To generate specific hydrogen-bonding interactions with a monoanionic substrate analogue, we have introduced a urea functional group into the active site by replacing arginine side chains with isosteric citrulline (Cit) residues. This design was based on the complementarity between the urea functionality of citrulline and the uncharged amide function of the substrate, as opposed to the guanidinium-carboxylate electrostatic interaction between the wild-type enzyme and the natural substrate. Indeed, the synthetic (Arg39Cit)4-OT analogue catalyzed the tautomerization of the non-natural monoamide-monoacid substrate while it was a poor catalyst for the natural diacid substrate. The specificity of (Arg39Cit)4-OT for the monoamide-monoacid substrate relative to that of the diacid substrate was found to be 740-fold greater than that of the wild-type enzyme for tautomerization of the non-natural substrate as compared with the natural one. The role of electrostatic interactions in the tautomerization of the monoamide-monoacid substrate was probed in detail with several other Arg to Cit analogues of this enzyme. This study has demonstrated that chemical manipulation of the functional groups within the active site of an enzyme can modify its catalytic activity and substrate specificity in a predictable way, suggesting that the incorporation of noncoded amino acids into proteins has great promise for the development of new enzymatic mechanisms and new binding interactions.     

Quantitative analysis of the effect of salt concentration on enzymatic catalysis.

Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt-rate profiles: k(cat)/K(M) = (k(cat)/K(M))(MAX)/[1+([Na+]/K[Na+])(n')], where (k(cat)/K(M))(MAX) is the physical limit of k(cat)/K(M), K(Na+) is the salt concentration at which k(cat)/K(M) = (k(cat)/K(M))(MAX)/2, and -n' is the slope of the linear region in a plot of log(k(cat)/K(M)) versus log [Na+]. The value of n' is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with k(cat)/K(M) values that can exceed 10(9) M(-1) s(-1). Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7, Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n'. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n'. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n', as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt-rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis.     

A new enzyme model for enantioselective esterases based on molecularly imprinted polymers

An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques. The enantiomerically pure phosphonic monoesters 4 L and 5 L were synthesized as stable transition-state analogues. They were used as templates connected by stoichiometric noncovalent interactions to two equivalents of the amidinium binding site monomer 1. After polymerization and removal of the template, the polymers were efficient catalysts for the hydrolysis of certain nonactivated amino acid phenylesters (2 L, 2 D, 3 L, 3 D) depending on the template used. Imprinted catalyst IP4 (imprinted with 4 L) enhanced the hydrolysis of the corresponding substrate 2 L by a factor of 325 relative to that of a buffered solution. Relative to a control polymer containing the same functionalities, prepared without template 4 L, the enhancement was still about 80-fold, showing the highest imprinting effect up to now. In cross-selectivity experiments a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template. Plots of initial velocities of the hydrolysis versus substrate concentration showed typical Michaelis-Menten kinetics with saturation behavior. From these curves, the Michaelis constant K(M) and the catalytic constant k(cat) can be calculated. The enantioselectivity shown in these values is most interesting. The ratio of the catalytic efficiency k(cat)/K(M), between the hydrolysis of 2 L- and 2 D-substrate with IP4, is 1.65. This enantioselectivity derives from both selective binding of the substrate (K(M)L/K(M)D=0.82), and from selective formation of the transition state (k(cat)L/k(cat)D=1.36). Thus, these catalysts give good catalysis as well as high imprinting and substrate selectivity. Strong competitive inhibition is caused by the template used in imprinting. This behavior is also quite similar to the behavior of natural enzymes, for which these catalysts are good models.     

Polyethylene imine derivatives ('synzymes') accelerate phosphate transfer in the absence of metal

The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.5 min(-1)) and up to 2340 turnovers per polymer molecule. Catalysis can be specifically and competitively inhibited by anionic and hydrophobic small molecules. The efficacy of catalysis is determined by the PEI derivatization pattern. The derivatization reagents exert a synergistic effect, i.e., their combinations increase catalysis by more than the sum of each single modification. The pH-rate profile for k(cat)/K(M) is bell shaped with a maximum at pH 7.85 and can be explained as a combination of two effects that both have to be operative for optimal activity: K(M) increases at high pH due to deprotonation of PEI amines that bind the anionic substrate and kcat decreases as the availability of hydroxide decreases at low pH. Thus, catalysis is based on substrate binding by positively charged amine groups and the presence of hydroxide ion in active sites in an environment that is tuned for efficient catalysis. Inhibition studies suggest that the basis of catalysis and multiple turnovers is differential molecular recognition of the doubly negatively charged transition state (over singly charged ground state and product): this contributes a factor of at least 5-10-fold to catalysis and product release.     

Molecular insight into the interaction between IFABP and PA by using MM-PBSA and alanine scanning methods

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with alanine-scanning mutagenesis is a very important tool for rational drug design. In this study, molecular dynamics (MD) and MM-PBSA were applied to calculate the binding free energy between the rat intestinal fatty acid binding protein (IFABP) and palmitic acid (PA) to gain insight to the interaction details. Equally spaced snapshots along the trajectory were chosen to perform the binding free energy calculation, which yields a result highly consistent with experimental value with a deviation of 0.4 kcal/mol. Computational alanine scanning was performed on the same set of snapshots by mutating the residues in IFABP to alanine and recomputing the DeltaDeltaG(binding). By postprocessing a single trajectory of the wild-type complex, the average unsigned error of our calculated DeltaDeltaG(binding) is below 1.5 kcal/mol for most of the alanine mutations of the noncharged residues (67% in total). To further investigate some particular mutants, three additional dynamical simulations of IFABP Arg126Ala, Arg106Ala, and Arg106Gln mutants were conducted. Recalculated binding free energies are well consistent with the experimental data. Moreover, the ambiguous role of Arg106 caused by the free energy change of the opposite sign when it is mutated to alanine and glutamine respectively is clarified both structurally and energetically. Typically, this can be attributed to the partial electrostatic compensation mainly from Arg56 and the obvious entropy gain in Arg106Ala mutant while not in Arg106Gln mutant. The presented structural model of IFABP-PA complex could be used to guide future studies.     

Steering the enzymatic activity of proteins by ionic liquids. A case study of the enzyme kinetics of yeast alcohol dehydrogenase

We explore ion-specific effects exerted by ionic liquids (ILs) on the enzyme kinetics of yeast alcohol dehydrogenase. The Michaelis-Menten reaction scheme is used to parameterize the observed kinetics in terms of the apparent dissociation constant of the substrate (Michaelis-Menten constant) K(M), the turnover number k(cat), which reflects the number of product molecules per enzyme molecule per second, and the enzymatic efficiency k(cat)/K(M) of the reaction. Results for fifteen salts are used to deduce Hofmeister anion and cation series. The ion rankings derived from K(M), k(cat) and k(cat)/K(M) differ markedly. Only the results for the enzymatic efficiency correspond to expectations from other phenomena, such as the thermal stability of native proteins. Anion variation has a significantly larger effect on the enzymatic efficiency than cation variation. All ILs decrease k(cat) relative to its value for the IL-free solution, thus driving enzyme deactivation. Enhancements of the enzymatic efficiency by some ions are founded in their effects on the Michaelis-Menten constant. The observed Hofmeister anion and cation series point toward hydrophobic interactions as an important factor controlling ion-specific effects on the enzymatic activity.     

Synthesis of potent CXCR4 inhibitors possessing low cytotoxicity and improved biostability based on T140 derivatives

A peptidic CXCR4 antagonist T140 efficiently blocks the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into target cells. In this study, a series of T140 derivatives, replacing the basic amino acid residues with Glu (D-Glu) and/or L-citrulline (Cit), were synthesized in order to reduce non-specific binding and cytotoxicity. Among them, TE14011 ([Cit6, D-Glu8]-T140 with the C-terminal amide) exhibited strong anti-HIV activity and low cytotoxicity. TE14011 was found to be stable in mouse serum, but unstable in rat liver homogenate due to the deletion of the N-terminal Arg1-Arg2-L-3-(2-naphthyl)alanine (Nal)3 residues from the parent peptide. N-Terminal acetylation of TE14011 led to the development of a novel lead compound, Ac-TE 14011, which possesses a high selectivity index as well as increased stability in serum and liver homogenate.     

Paramagnetic cobalt(II) as a probe for kinetic and NMR relaxation studies of phosphate binding and the catalytic mechanism of Streptomyces dinuclear aminopeptidase

Phosphate was proposed to be a bridging ligand in the structure 1xjo.pdb of Streptomyces dizinc aminopeptidase (sAP), which prompted further studies of phosphate binding to this enzyme. Phosphate inhibits sAP and its Co(2+)-substituted derivatives in a noncompetitive manner from pH 6.0 to 9.0, with strongest inhibition observed at lower pHs (K(i) = 0.6, 8.2, and 9.1 mM for ZnZn-, CoCo-, and CoZn-sAP, respectively, at pH 6.0), which indicates that phosphate does not compete with substrate binding to the dinuclear active site and that monobasic phosphate has a higher binding affinity. The inhibition K(i)-pH profiles for phosphate inhibition of both the native and the Co(2+)-substituted derivatives reveal a similar pK(a) around 7.0, reflecting that phosphate binding is not affected by the metal centers of different Lewis acidities. Modification of ZnZn- and CoCo-sAP with the arginine-specific reagent phenylglyoxal reveals a significant weakening in phosphate and substrate binding by showing approximately a 10-fold increase in the dissociation constant K(i) for phosphate binding and approximately 4-8-fold increase in K(m). The catalysis is also influenced by the modification as reflected by a significant decrease in k(cat) in both cases. Furthermore, phosphate and the transition-state inhibitor 1-aminobutyl phosphonate can protect arginine from the modification, strongly suggesting that Arg202 near the active site is involved in phosphate binding and in stabilizing the transition state. The effect on (31)P NMR relaxation of phosphate caused by the paramagnetic metal center in Co(2+)-substituted derivatives of sAP has been measured, which reveals that only one phosphate is bound to sAP with the Co(2+)-(31)P distance in the range of 4.1-4.3 A. The (1)H NMR relaxation of the bulk water signal in the CoCo-sAP sample remains unchanged in the presence of phosphate, further indicating that phosphate may not bind to the active-site metals to displace any metal-bound water/hydroxide. These results strongly support that the phosphate binding site is Arg202 and that this residue plays an important role in the action of sAP.     

Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis

Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg(2+)-binding domains led in most cases to reductions in both k(cat) and k(cat)/K(m) as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in k(cat) that was offset by a 50-fold decrease in K(m), resulting in a relatively minor 2-fold decrease in catalytic efficiency, k(cat)/K(m). The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4).     

Phosphatase-like activity, DNA binding, DNA hydrolysis, anticancer and lactate dehydrogenase inhibition activity promoting by a new bis-phenanthroline dicopper(II) complex

A new bis-phenanthroline dicopper(II) complex has been synthesized and characterized by elemental analysis and spectroscopic methods. The molecular structure of the dinuclear Cu(II) complex [Cu(2)(μ-CH(3)COO)(μ-H(2)O)(μ-OH)(phen)(2)](2+) (phen = 1,10-phenanthroline) (1) was determined by single crystal X-ray diffraction technique. The coordination environment around each Cu(II) ion in complex 1 can be described as slightly distorted square pyramidal geometry. The distance between the CuCu centers in the complex is found to be 2.987 ?. The electronic, redox, phosphate hydrolysis, DNA binding and DNA cleavage have been studied. The antiproliferative effect of complex 1 was confirmed by the lactate dehydrogenase (LDH) enzyme level in MCF-7 cancer cell lysate and content media. The dicopper(II) complex inhibited the LDH enzyme as well as the growth of the human breast cancer MCF7 cell line at an IC(50) value of 0.011 μg ml(-1). The results strongly suggest that complex 1 is a good cancer therapeutic agent. Electrochemical studies of complex 1 showed an irreversible, followed by a quasi-reversible, one electron reduction processes between -0.20 to -0.8 V. Michaelis-Menten kinetic parameters for the hydrolysis of 4-nitrophenyl phosphate by complex 1 are k(cat) = 3.56 × 10(-2) s(-1) and K(M) = 4.3 × 10(-2) M. Complex 1 shows good binding propensity to calf thymus DNA, with a binding constant value of 1.3 (±0.13) × 10(5) M(-1) (s = 2.1). The size of the binding site and viscosity data suggest a DNA intercalative binding nature of the complex. Complex 1 shows efficient hydrolytic cleavage of supercoiled pBR322-DNA in the dark and in the absence of any external reagents, as demonstrated by the T4 ligase experiment. The pseudo-Michaelis-Menten kinetic parameters for DNA hydrolysis by complex 1 are k(cat) = 1.27 ± 0.4 h(-1) and K(M) = 7.7 × 10(-2) M.     

Beta-D-xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

Beta-D-Xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D: -xylooligosaccharides to D-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-beta-D-xylopyranoside (4NPX), 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), and 1,4-beta-D-xylobiose (X2) was determined on and off (k (non)) the enzyme at pH 5.3, which lies in the pH-independent region for k (cat) and k (non). Rate enhancements (k (cat)/k (non)) for 4NPX, 4NPA, and X2 are 4.3 x 10(11), 2.4 x 10(9), and 3.7 x 10(12), respectively, at 25 degrees C and increase with decreasing temperature. Relative parameters k (cat) (4NPX)/k (cat) (4NPA), k (cat) (4NPX)/k (cat) (X2), and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(X2) increase and (k (cat)/K (m))(4NPX)/(k (cat)/K (m))(4NPA), (1/K (m))(4NPX)/(1/K (m))(4NPA), and (1/K (m))(4NPX)/(1/K (m))(X2) decrease with increasing temperature.     

Low-energy collisionally activated decomposition and structural characterization of cyclic heptapeptide microcystins by electrospray ionization mass spectrometry

Characteristics of electrospray ionization mass spectrometry/collision-induced dissociation (ESIMS/CID) mass spectra of microcystins, cyanobacterial cyclic heptapeptide hepatoxins, were examined. The collision conditions showed remarkable effects on the quality of the CID mass spectra, which were divided into three patterns according to the number of Arg residues. A characteristic cleavage reaction and neutral losses of MeOH, NH3 and guanidine group(s) from the (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4 E,6E-dienoic acid (Adda) and Arg residues were observed in the ESI and ESIMS/CID mass spectra, suggesting the most probable protonation sites in [M + H]+ and [M + 2H]2+ ions of microcystins. Microcystins with no Arg residue showed only [M + H]+ ions with a proton reacting at the methoxyl group in the Adda residue, and the ESIMS/CID/MS data revealed their structures unambiguously. The protonation site in [M + H]+ ions of microcystins with Arg residue(s) was the guanidine group. The [M + 2H]2+ ions of microcystins possessing one Arg residue had one proton on the Arg residue and probably another proton on the Adda residue, while the [M + 2H]2+ ions of microcystins having two Arg residues showed protonation at both Arg residues and the ESIMS/CID/MS data assigned their sequences. Structures of microcystins possessing one Arg residue can be assigned by ESIMS/CID/MS of [M + H]+ ions combined with those of [M + 2H]2+ ions.     

Furcatin 水解酶的三维结构模建和与furcatin 的对接研究

利用同源模建和动力学模拟方法，模建了furcatin水解酶（FH）的三维结构．并在这基础上，分析了活性位点的组成和结构．研究了furcatin与FH的对接．结果表明，Ser84，Arg146，Thr189，Thr234和Gly372在复合物的形成过程中起重要的作用．其中，Ser84，Argl46和Thr189是在FH的活性口袋的二糖部分的亚单位一1中重要的氨基酸，Thr234和Gly372是亚单位-2中重要的氨基酸．     

Studying enzyme binding specificity in acetylcholinesterase using a combined molecular dynamics and multiple docking approach

A combined molecular dynamics simulation and multiple ligand docking approach is applied to study the binding specificity of acetylcholinesterase (AChE) with its natural substrate acetylcholine (ACh), a family of substrate analogues, and choline. Calculated docking energies are well correlated to experimental k(cat)/K(M) values, as well as to experimental binding affinities of a related series of TMTFA inhibitors. The "esteratic" and "anionic" subsites are found to act together to achieve substrate binding specificity. We find that the presence of ACh in the active site of AChE not only stabilizes the setup of the catalytic triad but also tightens both subsites to achieve better binding. The docking energy gained from this induced fit is 0.7 kcal/mol for ACh. For the binding of the substrate tailgroup to the anionic subsite, both the size and the positive charge of the tailgroup are important. The removal of the positive charge leads to a weaker binding of 1 kcal/mol loss in docking energy. Substituting each tail methyl group with hydrogen results in both an incremental loss in docking energy and also a decrease in the percentage of structures docked in the active site correctly set up for catalysis.     

Fundamental reaction mechanism for cocaine hydrolysis in human butyrylcholinesterase

Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.     

Synthesis and esterolytic activity of catalytic peptide dendrimers

Peptide dendrimers were prepared by solid-phase peptide synthesis. Monomeric dendrimers were first obtained by assembly of a hexapeptide sequence containing alternate standard alpha-amino acids with diamino acids as branching units. The monomeric dendrimers were then dimerized by disulfide-bridge formation at the core cysteine. The synthetic strategy is compatible with functional amino acids and different diamino acid branching units. Peptide dendrimers composed of the catalytic triad amino acids histidine, aspartate, and serine catalyzed the hydrolysis of N-methylquinolinium salts when the histidine residues were placed at the outermost position. The dendrimer-catalyzed hydrolysis of 7-isobutyryl-N-methylquinolinium followed saturation kinetics with a rate constant of catalysis/rate constant without catalysis (k(cat)/k(uncat)) value of 3350 and a rate constant of catalysis/Michaelis constant (k(cat)/K(M)) value 350-fold larger than the second-order rate constant of the 4-methylimidazole-catalyzed reaction; this corresponds to a 40-fold rate enhancement per histidine side chain. Catalysis can be attributed to the presence of histidine residues at the surface of the dendrimers.     

Analysis of the importance of the metallo-beta-lactamase active site loop in substrate binding and catalysis

The role of the mobile loop comprising residues 60-66 in metallo-beta-lactamases has been studied by site-directed mutagenesis, determination of kinetic parameters for six substrates and two inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and molecular modeling. The W64A mutation was performed in IMP-1 and BcII (after replacement of the BcII 60-66 peptide by that of IMP-1) and always resulted in increased K(i) and K(m) and decreased k(cat)/K(m) values, an effect reinforced by complete deletion of the loop. k(cat) values were, by contrast, much more diversely affected, indicating that the loop does not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand is also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications.     

Remarkable remote chiral recognition in a reaction mediated by a catalytic antibody

The crystal structures of catalytic antibody D2.3 Fab with the two enantiomers, 7D and 7L, which represent transition state analogues for the hydrolysis of the corresponding esters, 6D and 6L, were determined to better understand remarkable reactivity differences: the L-ester displayed significantly tighter binding (K(M)) and increased catalytic activity (k(cat)) with D2.3, even though the chiral center is 7 bonds distant from the reaction center. Surprisingly, the electron densities of the liganded phosphonates, 7D and 7L, within the D2.3 binding/reaction site were essentially identical, highlighting the subtle influences of protein interactions on chemical behavior.     

基于分子模拟方法预测PIP2在双孔钾通道TREK-1上结合位点的研究

磷脂酰肌醇4,5-二磷酸酯(PIP₂)是一类分布在质膜内层的信号磷脂分子, 对钾、 钠和氯等离子通道和转运蛋白等多种跨膜蛋白具有调节作用. TREK-1是一类重要的背景钾通道, 受温度、 机械拉伸及胞内pH等多种因素调节, PIP₂在特定浓度范围内可激活TREK-1通道, 在内面向外膜片钳记录TREK-1通道电流中使用PIP₂抗结剂(如多聚赖氨酸)可导致TREK-1通道关闭. 利用分子对接和全原子分子动力学模拟探索了PIP₂与双孔钾通道TREK-1的相互作用. 分子对接计算结果表明, PIP₂在TREK-1通道上有两个可能的结合位点. 进一步的分子动力学模拟和均力势(PMF)计算结果表明, 其中位于螺旋M4和螺旋M1的位点可能是PIP₂激活TREK-1的优先结合位点. 模拟展示了PIP₂与TREK-1结合的可能构象. PIP₂的肌醇头部磷酸根与位于M1和M4上的碱性残基K45, K304和R311形成稳定盐桥; M1螺旋上的一系列疏水残基对稳定PIP₂的脂肪长链具有关键作用.     

The first branching point in porphyrin biosynthesis: a systematic docking, molecular dynamics and quantum mechanical/molecular mechanical study of substrate binding and mechanism of uroporphyrinogen-III decarboxylase

In humans, uroporphyrinogen decarboxylase is intimately involved in the synthesis of heme, where the decarboxylation of the uroporphyrinogen-III occurs in a single catalytic site. Several variants of the mechanistic proposal exist; however, the exact mechanism is still debated. Thus, using an ONIOM quantum mechanical/molecular mechanical approach, the mechanism by which uroporphyrinogen decarboxylase decarboxylates ring D of uroporphyrinogen-III has been investigated. From the study performed, it was found that both Arg37 and Arg50 are essential in the decarboxylation of ring D, where experimentally both have been shown to be critical to the catalytic behavior of the enzyme. Overall, the reaction was found to have a barrier of 10.3 kcal mol(-1) at 298.15 K. The rate-limiting step was found to be the initial proton transfer from Arg37 to the substrate before the decarboxylation. In addition, it has been found that several key interactions exist between the substrate carboxylate groups and backbone amides of various active site residues as well as several other functional groups.