Catalytic Mechanism of Cruzain from Trypanosoma cruzi As Determined from Solvent Kinetic Isotope Effects of Steady-State and Pre-Steady-State Kinetics

Biochemistry ◽  
2018 ◽  
Vol 57 (22) ◽  
pp. 3176-3190 ◽  
Author(s):  
Xiang Zhai ◽  
Thomas D. Meek
Keyword(s):  
Steady State ◽  
Trypanosoma Cruzi ◽  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope ◽  
Steady State Kinetics

Catalytic Mechanism of Scytalone Dehydratase:  Site-Directed Mutagenisis, Kinetic Isotope Effects, and Alternate Substrates

Biochemistry ◽  
1999 ◽  
Vol 38 (19) ◽  
pp. 6012-6024 ◽  
Author(s):  
Gregory S. Basarab ◽  
James J. Steffens ◽  
Zdzislaw Wawrzak ◽  
Rand S. Schwartz ◽  
Tomas Lundqvist ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope ◽  
Scytalone Dehydratase

scholarly journals Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

2016 ◽  
Vol 113 (52) ◽  
pp. E8369-E8378 ◽  
Author(s):  
Joshua A. Linscott ◽  
Kanishk Kapilashrami ◽  
Zhen Wang ◽  
Chamara Senevirathne ◽  
Ian R. Bothwell ◽  
...  
Keyword(s):  
Transition State ◽  
Cell Cycle Progression ◽  
Cancer Metastasis ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Substrate Complex ◽  
Flight Mass Spectrometry ◽  
Kinetic Isotope ◽  
Steady State Kinetics ◽  
Lysine Methyltransferase

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)–PKMT–substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactorS-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic13C KIE of 1.04, an inverse intrinsic α-secondary CD3KIE of 0.90, and a small but statistically significant inverse CD3BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35–2.40 Å and 2.00–2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analogSe-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.

The pH dependence of xanthine oxidase catalysis in basic solution

1980 ◽  
Vol 58 (5) ◽  
pp. 394-398 ◽  
Author(s):  
John W. Bunting ◽  
Keith R. Laderoute ◽  
Donald J. Norris
Keyword(s):  
Xanthine Oxidase ◽  
Isotope Effects ◽  
Ph Dependence ◽  
Kinetic Isotope Effects ◽  
Similar Rate ◽  
Basic Solution ◽  
Kinetic Isotope ◽  
Steady State Kinetics ◽  
Pyridinium Cations ◽  
Kinetics Of

The steady-state kinetics of the oxidation of the following six heteroaromatic substrates by xanthine oxidase have been investigated over the range pH 9.0–11.1 at 25 °C, ionic strength 0.1: 1-methylquinolinium, 6-methoxy-1-methylquinolinium, 1-methylnicotinamide, 3-acetyl-1-methylpyridinium, and 1-(4-methoxyphenyl)pyridinium cations and 1-methylnicotinate zwitterion. For the first four of these species, kc and Km were evaluated as a function of pH while only kc/Km was accessible in the latter two cases. Where available, kc is pH independent, whereas plots of log (kc/Km) vs. pH are linear with slopes in the range 0.54–1.17.The rates of enzymic oxidation of the 1-methylquinolinium cation and its 2-deuterio derivative were investigated and kinetic isotope effects were calculated at pH 9.8 and 10.6: kcH/kcD = 1.7 and KmH/KmD = 0.4 at each pH. Detailed comparisons of the oxidation of heteroaromatic cations and xanthine-derived substrates indicate that similar rate-determining steps control the enzymic oxidations of these two classes of substrate.

scholarly journals Kinetic isotope effects and ligand binding in PQQ-dependent methanol dehydrogenase

2005 ◽  
Vol 388 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Parvinder HOTHI ◽  
Michael J. SUTCLIFFE ◽  
Nigel S. SCRUTTON
Keyword(s):  
Steady State ◽  
Active Site ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Competitive Binding ◽  
Catalytic Site ◽  
Methanol Dehydrogenase ◽  
Ammonium Concentration ◽  
Steric Interaction ◽  
Kinetic Isotope

The reaction of PQQ (2,7,9-tricarboxypyrroloquinoline quinone)-dependent MDH (methanol dehydrogenase) from Methylophilus methylotrophus has been studied under steady-state conditions in the presence of an alternative activator [GEE (glycine ethyl ester)] and compared with similar reactions performed with ammonium (used more generally as an activator in steady-state analysis of MDH). Studies of initial velocity with methanol (protiated methanol, C1H3O1H) and [2H]methanol (deuteriated methanol, C2H3O2H) as substrate, performed with different concentrations of GEE and PES (phenazine ethosulphate), indicate competitive binding effects for substrate and PES on the stimulation and inhibition of enzyme activity by GEE. GEE is more effective at stimulating activity than ammonium at low concentrations, suggesting tighter binding of GEE to the active site. Inhibition of activity at high GEE concentration is less pronounced than at high ammonium concentration. This suggests a close spatial relationship between the stimulatory (KS) and inhibitory (KI) binding sites in that binding of GEE to the KS site sterically impairs the binding of GEE to the KI site. The binding of GEE is also competitive with the binding of PES, and GEE is more effective than ammonium in competing with PES. This competitive binding of GEE and PES lowers the effective concentration of PES at the site competent for electron transfer. Accordingly, the oxidative half-reaction, which is second-order with respect to PES concentration, is more rate-limiting in steady-state turnover with GEE than with ammonium. The smaller methanol C-1H/C-2H kinetic isotope effects observed with GEE are consistent with a larger contribution made by the oxidative half-reaction to rate limitation. Cyanide is much less effective at suppressing ‘endogenous’ activity in the presence of GEE than with ammonium, which is attributed to impaired binding of cyanide to the catalytic site through steric interaction with GEE bound at the KS site. The kinetic model developed previously for reactions of MDH with ammonium [Hothi, Basran, Sutcliffe and Scrutton (2003) Biochemistry 42, 3966–3978] is consistent with data obtained with GEE, although a more detailed structural interpretation is given here. Molecular-modelling studies rationalize the kinetic observations in terms of a complex binding scenario at the molecular level involving two spatially distinct inhibitory sites (KI and KI′). The KI′ site caps the entrance to the active site and is interpreted as the PES binding site. The KI site is adjacent to, and, for GEE, overlaps with, the KS site, and is located in the active-site cavity close to the PQQ cofactor and the catalytic site for methanol oxidation.

scholarly journals Catalytic mechanism of a family 3 β-glucosidase and mutagenesis study on residue Asp-247

2001 ◽  
Vol 355 (3) ◽  
pp. 835-840 ◽  
Author(s):  
Yaw-Kuen LI ◽  
Jiunly CHIR ◽  
Fong-Yi CHEN
Keyword(s):  
Amino Acid ◽  
Essential Amino Acid ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Kinetic Isotope

A family 3 β-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the kcat values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl–enzyme intermediate. The large Bronsted constant (β =-0.85) for the leaving-group-dependent portion (pKa of leaving phenols > 7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl β-D-glucopyanoside, o-nitrophenyl β-D-glucopyanoside and p-cyanophenyl β-D-glucopyanoside as substrates were 1.17±0.02, 1.19±0.02 and 1.04±0.02 respectively. These results support an SN1-like mechanism for the deglucosylation step and an SN2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (kcat/Km) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

scholarly journals The catalytic mechanism of the Suzuki-Miyaura reaction

2021 ◽  
Author(s):  
Juliet macharia ◽  
Chetan Joshi ◽  
Joseph Izzo ◽  
Victor Wambua ◽  
Sungjin Kim ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Palladium Complex ◽  
Isotope Effects ◽  
Oxidative Addition ◽  
Kinetic Isotope Effects ◽  
Boronic Acids ◽  
Aryl Halides ◽  
Kinetic Isotope ◽  
Aryl Bromides ◽  
Insight Into

Abstract: Experimental and theoretical 13C kinetic isotope effects are utilized to obtain atomistic insight into the catalytic mechanism of the Pd(PPh3)4 catalyzed Suzuki-Miyaura reaction of aryl halides and aryl boronic acids. Under catalytic conditions, we establish that oxidative addition of aryl bromides occurs to a 12-electron monoligated palladium complex (Pd(PPh3)). For aryl iodides, the first irreversible step in the catalytic cycle precedes oxidative addition and is shown to be binding of the iodoarene to Pd(PPh3). Our results suggest that the commonly proposed oxidative addition to the 14-electron Pd(PPh3)2 complex can occur only in the presence of excess added ligand or under stoichiometric conditions. The transmetalation step, under catalytic conditions, is shown to proceed via a tetracoordinate boronate (8B4) intermediate with a Pd-O-B linkage.

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