scholarly journals On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase

2003 ◽  
Vol 375 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Mario KLIMACEK ◽  
Kathryn L. KAVANAGH ◽  
David K. WILSON ◽  
Bernd NIDETZKY
Keyword(s):  
Pseudomonas Fluorescens ◽  
Time Course ◽  
Isotope Effects ◽  
Secondary Alcohol ◽  
Molecular Size ◽  
Order Rate Constant ◽  
Site Directed Mutagenesis ◽  
Linear Slope ◽  
Solvent Isotope ◽  
Relationship Of

X-ray structure of the Pseudomonas fluorescens mannitol 2-dehydrogenase ternary complex with NAD+ and d-mannitol suggests that Lys-295 provides catalytic base assistance to secondary alcohol group oxidation. We have replaced Lys-295 by site-directed mutagenesis with alanine or methionine and evaluated the catalytic significance of side-chain substitution by kinetic analysis of restoration of activity with external amines, and from pH and solvent isotope effects on the reaction catalysed by K295A (Lys-295→Ala mutant). K295A and K295M (Lys-295→Met mutants) show 3×104- and 2×106-fold lower turnover numbers respectively for d-mannitol oxidation (kcatO) at pH 10.0 than the wild-type. The second-order rate constant for non-covalent rescue of activity (kB) by free methylamine base is 31 M−1·s−1 for K295A, but only 0.021 M−1·s−1 for K295M. A Brønsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope β=0.66±0.16; r2=0.99) for K295A-catalysed d-mannitol oxidation at pH 10.0. The kcatO values of K295A in H2O and 2H2O are linearly dependent on [OL−] in the pL range 7.5–10.5 (where L is 1H or 2H). The solvent isotope effect on kcatO is 0.69. The time course of d-fructose reduction by K295A at pH 8.2 displays a pre-steady-state burst of NADH consumption. These data support a mechanism in which the ε-NH2 group of Lys-295 participates in an obligatory pH-dependent, pre-catalytic equilibrium which may control alcohol/alkoxide equilibration of enzyme-bound d-mannitol and activates the C2 atom for subsequent catalytic oxidation by NAD+.

scholarly journals A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens

2002 ◽  
Vol 367 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Mario KLIMACEK ◽  
Bernd NIDETZKY
Keyword(s):  
Pseudomonas Fluorescens ◽  
Isotope Effects ◽  
Wild Type ◽  
Solvent Isotope Effect ◽  
General Base ◽  
Kinetic Isotope ◽  
Solvent Isotope ◽  
Auxiliary Role ◽  
Long Chain

Lys-295, Asn-300 and His-303 of d-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD+-dependent oxidation of d-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for d-fructose reduction by wild-type and mutants at pH10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11±1 on kcat for mannitol oxidation by K295A at pH(2H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa4-Asn-Xaa2-His (where ‘Xaa’ denotes ‘any amino acid').

scholarly journals Interaction of aspartate aminotransferase with mercurochrome. Relationship of an exposed thiol group of the enzyme to the active centre

1977 ◽  
Vol 167 (1) ◽  
pp. 53-63 ◽  
Author(s):  
T G Kalogerakos ◽  
N G Oikonomakos ◽  
C G Dimitropoulos ◽  
I A Karni-katsadima ◽  
A E Evangelopoulos
Keyword(s):  
Time Course ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Second Order ◽  
Aspartate Transaminase ◽  
Difference Spectra ◽  
Active Centre ◽  
Order Rate ◽  
Modified Enzyme ◽  
Relationship Of

Mercurochrome strongly inhibits aspartate transaminase and 2,3-dicarboxyethylated aspartate transaminase. The native enzyme exhibits a biphasic time-course of inactivation by mercurochrome with second-order rate constants 1.62 × 10(4) M-1 - min-1 and 2.15 × 10(3) M-1 - min-1, whereas the modified enzyme is inactivated more slowly (second-order rate constant 6.1 × 10(2) M-1 - min-1) under the same conditions. The inhibitor inactivates native and modified enzyme in the absence as well as in the presence of substrates. Mercurochrome-transaminase interaction is accompanied by a red shift in the absorption maximum of the fluorochrome of about 10 nm. Difference spectra of the mercurochrome-enzyme system versus mercurochrome, compared with analogous spectra of mercurochrome-ethanol, revealed that the spectral shifts recorded during mercurochrome-transaminase interaction are similar to those that occur when mercurochrome is dissolved in non-polar solvents. Studies of mercurochrome complexes with native or modified transaminase, isolated by chromatography on Sephadex G-25, revealed that native transaminase is able to conjugate with four mercurochrome molecules per molecule, but the modified enzyme is able to conjugate with only two mercurochrome molecules per molecule.

scholarly journals Epoxidation and late-stage C-H functionalization by P450 TamI is mediated by variant heme-iron oxidizing species

2021 ◽  
Author(s):  
Rosa V. Espinoza ◽  
Mark A. Maskeri ◽  
Aneta Turlik ◽  
Anjanay Nangia ◽  
Yogan Khatri ◽  
...  
Keyword(s):  
Density Functional ◽  
Isotope Effects ◽  
Heme Iron ◽  
Site Directed Mutagenesis ◽  
Compound I ◽  
Iron Species ◽  
Epoxide Opening ◽  
Catalytically Active ◽  
Common Substrate ◽  
Solvent Isotope

P450-catalyzed hydroxylation reactions are well understood mechanistically including the identity of the active oxidizing species. However, the catalytically active heme-iron species in P450 iterative oxidation cascades that involve mechanistically divergent pathways and distinct carbon atoms within a common substrate remains unexplored. Recently, we reported the enzymatic synthesis of tri-functionalized tirandamycin O (9) and O’ (10) using a bacterial P450 TamI variant and developed mechanistic hypotheses to explore their formation. Here, we report the ability of bacterial P450 TamI L295A to shift between different oxidizing species as it catalyzes the sequential epoxidation, hydroxylation and radical-catalyzed epoxide-opening cascade to create new tirandamycin antibiotics. We also provide evidence that the TamI peroxo-iron species could be a viable catalyst to enable nucleophilic epoxide opening in the absence of iron-oxo Compound I. Using site-directed mutagenesis, kinetic solvent isotope effects, artificial oxygen surrogates, end-point assays, and density functional theory (DFT) calculations, we provide new insights into the active oxidant species that P450 TamI employs to introduce its unique pattern of oxidative decorations.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

scholarly journals Rate and Product Studies with 1-Adamantyl Chlorothioformate under Solvolytic Conditions

2021 ◽  
Vol 22 (14) ◽  
pp. 7394
Author(s):  
Kyoung Ho Park ◽  
Mi Hye Seong ◽  
Jin Burm Kyong ◽  
Dennis N. Kevill
Keyword(s):  
Rate Constants ◽  
Isotope Effects ◽  
Ion Pairs ◽  
Carbonyl Sulfide ◽  
Activation Parameters ◽  
Chloride Ions ◽  
Reaction Channels ◽  
Decomposition Pathway ◽  
Solvent Isotope ◽  
Solvent Isotope Effects

A study was carried out on the solvolysis of 1-adamantyl chlorothioformate (1-AdSCOCl, 1) in hydroxylic solvents. The rate constants of the solvolysis of 1 were well correlated using the Grunwald–Winstein equation in all of the 20 solvents (R = 0.985). The solvolyses of 1 were analyzed as the following two competing reactions: the solvolysis ionization pathway through the intermediate (1-AdSCO)+ (carboxylium ion) stabilized by the loss of chloride ions due to nucleophilic solvation and the solvolysis–decomposition pathway through the intermediate 1-Ad+Cl− ion pairs (carbocation) with the loss of carbonyl sulfide. In addition, the rate constants (kexp) for the solvolysis of 1 were separated into k1-Ad+Cl− and k1-AdSCO+Cl− through a product study and applied to the Grunwald–Winstein equation to obtain the sensitivity (m-value) to change in solvent ionizing power. For binary hydroxylic solvents, the selectivities (S) for the formation of solvolysis products were very similar to those of the 1-adamantyl derivatives discussed previously. The kinetic solvent isotope effects (KSIEs), salt effects and activation parameters for the solvolyses of 1 were also determined. These observations are compared with those previously reported for the solvolyses of 1-adamantyl chloroformate (1-AdOCOCl, 2). The reasons for change in reaction channels are discussed in terms of the gas-phase stabilities of acylium ions calculated using Gaussian 03.

scholarly journals Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence

1991 ◽  
Vol 275 (3) ◽  
pp. 767-773 ◽  
Author(s):  
Y K Li ◽  
J Boggaram ◽  
L D Byers
Keyword(s):  
Isotope Effect ◽  
Rate Constant ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Acidic Group ◽  
Irreversible Inhibitors ◽  
Effects Of Ph ◽  
Solvent Isotope ◽  
Bell Shaped Curve

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.

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