scholarly journals Kinetic isotope effects of peptidylglycine α-hydroxylating mono-oxygenase reaction

1998 ◽  
Vol 336 (1) ◽  
pp. 131-137 ◽  
Author(s):  
Kenichi TAKAHASHI ◽  
Tetsuo ONAMI ◽  
Masato NOGUCHI
Keyword(s):  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Tracer Method ◽  
Deuterium Isotope Effect ◽  
Critical Determinant ◽  
Product Release ◽  
Kinetic Isotope ◽  
Hydroxylation Reaction ◽  
Double Label ◽  
Rate Limiting

Many bioactive polypeptides or neuropeptides possess a C-terminal α-amide group as a critical determinant for their optimal bioactivities. The amide functions are introduced by the sequential actions of peptidylglycine α-hydroxylating mono-oxygenase (PHM; EC 1.14.17.3) and peptidylamidoglycollate lyase (PAL; EC 4.3.2.5) from their glycine-extended precursors. In the present study we examined the kinetic isotope effects of the frog PHM reaction by competitive and non-competitive approaches. In the competitive approach we employed the double-label tracer method with d-Tyr-[U-14C]Val-Gly, d-Tyr-[3,4-3H]Val-[2,2-2H2]-Gly, and d-Tyr-Val-(R,S)[2-3H]Gly as substrates, and we determined the deuterium and tritium effects on Vmax/Km as 1.625±0.041 (mean±S.D.) and 2.71±0.16 (mean±S.D.), respectively. The intrinsic deuterium isotope effect (Dk) on the glycine hydroxylation reaction was estimated to be 6.5–10.0 (mean 8.1) by the method of Northrop [Northrop (1975) Biochemistry 14, 2644–2651]. In the non-competitive approach with N,N-dimethyl-1,4-phenylenediamine as a reductant, however, the deuterium effect on Vmax (DV) was approximately unity, although the deuterium effect on Vmax/Km (DV/K) was comparable to that obtained by the competitive approach. These results indicated that DV was completely masked by the presence of one or more steps much slower than the glycine hydroxylation step and that DV/K was diminished from Dk by a large forward commitment to catalysis. The addition of PAL, however, increased the apparent DV from 1.0 to 1.2, implying that the product release step was greatly accelerated by PAL. These results suggest that the product release is rate-limiting in the overall PHM reaction. The large Dk indicated that the glycine hydroxylation catalysed by PHM might proceed in a stepwise mechanism similar to that proposed for the dopamine β-hydroxylase reaction [Miller and Klinman (1983) Biochemistry 22, 3091–3096].

DEUTERIUM KINETIC ISOTOPE EFFECTS: V TEMPERATURE DEPENDENCE OF β-DEUTERIUM EFFECTS IN WATER SOLVOLYSIS OF ISOPROPYL COMPOUNDS

1961 ◽  
Vol 39 (10) ◽  
pp. 1989-1994 ◽  
Author(s):  
K. T. Leffek ◽  
R. E. Robertson ◽  
S. E. Sugamori
Keyword(s):  
Temperature Dependence ◽  
Isotope Effect ◽  
Isotope Effects ◽  
Zero Point ◽  
Kinetic Isotope Effects ◽  
Methyl Groups ◽  
Deuterium Isotope Effect ◽  
Point Energy ◽  
Kinetic Isotope ◽  
Enthalpy Of Activation

The secondary β-deuterium isotope effect (kH/kD) has been measured over a range of temperature for the water solvolysis reactions of isopropyl methanesulphonate, p-toluenesulphonate, and bromide. In these cases the isotope effect is due to a difference in entropies of activation of the isotopic analogues rather than a difference in the enthalpies of activation. It is suggested that the observed isotope effect is due to internal rotational effects of the methyl groups in the isopropyl radical, and the lack of an isotope effect on the enthalpy of activation is accounted for by a cancellation of an effect from this source and one from zero-point energy.

Unequal primary kinetic isotope effects in the base-catalysed H–D exchange of diastereotopic protons

1980 ◽  
Vol 58 (1) ◽  
pp. 72-78 ◽  
Author(s):  
Robert R. Fraser ◽  
Philippe J. Champagne
Keyword(s):  
Transition State ◽  
Exchange Reaction ◽  
Isotope Effect ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Deuterium Isotope Effect ◽  
Kinetic Isotope

Primary kinetic isotope effects have been measured for the base-catalyzed exchange reaction of 4′,1″-dimethyl-1,2,3,4-dibenzcyclohepta-1,3-diene-6-one, 1. It was found that the isotope effects kH/kT and kD/kT for the faster exchanging protons (13.6 and 3.8 respectively) are significantly larger than the corresponding values for the slower exchanging protons (4.6 and 1.6 respectively). These differences could result from truly unequal isotope effects due to transition state differences or intrusion of a second pathway for exchange of the less reactive proton in the dedeuteration reaction. The data appear to support the latter interpretation. The secondary deuterium isotope effect was found to be 1.18.

Experimental and Theoretical Kinetic Isotope Effects for Asymmetric Dihydroxylation. Evidence Supporting a Rate-Limiting “(3 + 2)” Cycloaddition

1997 ◽  
Vol 119 (41) ◽  
pp. 9907-9908 ◽  
Author(s):  
Albert J. DelMonte ◽  
Jan Haller ◽  
K. N. Houk ◽  
K. Barry Sharpless ◽  
Daniel A. Singleton ◽  
...  
Keyword(s):  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Asymmetric Dihydroxylation ◽  
Kinetic Isotope ◽  
Rate Limiting

scholarly journals Comment on “Topography of the free energy landscape of Claisen–Schmidt condensation: solvent and temperature effects on the rate-controlling step” by N. D. Coutinho, H. G. Machado, V. H. Carvalho-Silva and W. A. da Silva, Phys. Chem. Chem. Phys., 2021, 23, 6738

2021 ◽  
Vol 23 (38) ◽  
pp. 22199-22201
Author(s):  
Charles L. Perrin
Keyword(s):  
Temperature Effects ◽  
Isotope Effects ◽  
Energy Landscape ◽  
Kinetic Isotope Effects ◽  
Chem Phys ◽  
Kinetic Isotope ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Rate Controlling Step

The referenced article in PCCP presents calculations of solvent kinetic isotope effects that indicate that the rate-limiting step in base-catalyzed chalcone formation in aqueous solution becomes the second enolization.

scholarly journals Use of pH and Kinetic Isotope Effects To Establish Chemistry as Rate-Limiting in Oxidation of a Peptide Substrate by LSD1

Biochemistry ◽  
2009 ◽  
Vol 48 (23) ◽  
pp. 5440-5445 ◽  
Author(s):  
Helena Gaweska ◽  
Michelle Henderson Pozzi ◽  
Dawn M. Z. Schmidt ◽  
Dewey G. McCafferty ◽  
Paul F. Fitzpatrick
Keyword(s):  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Peptide Substrate ◽  
Kinetic Isotope ◽  
Rate Limiting

scholarly journals The effect of glycerol on the activity of β-glucosidase from Botryodiplodia theobromae Pat

1988 ◽  
Vol 254 (1) ◽  
pp. 73-76 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Rate Equations ◽  
Glycerol Concentration ◽  
Large Excess ◽  
Reaction Sequence ◽  
Botryodiplodia Theobromae ◽  
Kinetic Isotope ◽  
Beta Glucosidase ◽  
Rate Limiting

1. In the activity of the high-Mr beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. on o-nitrophenyl beta-D-glucopyranoside as substrate, both Vmax. and Km increased non-linearly with increasing concentration of glycerol, and the Vmax./Km(app.) ratio decreased non-linearly with increasing concentration of glycerol. 2. No increase in rate was observed with phenyl beta-D-glucopyranoside as substrate in the presence of up to 250 mM-glycerol, indicating that glucosylation is rate-limiting with this substrate. 3. With o-nitrophenyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrates, kappa cat. values of 793.7 s-1, 62.8 s-1 and 5.4 s-1 respectively were calculated. 4. With o-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrate, alpha-deuterium kinetic isotope effects of 1.9 +/- 0.03 and 1.01 +/- 0.01 respectively were found; in the presence of 200 mM-glycerol the values were 1.21 +/- 0.03 and 1.02 +/- 0.01 respectively. 5. In the presence of a large excess of o-nitrophenyl beta-D-glucopyranoside [(S] = 35.7 Km), the amount of o-nitrophenol and also of the transglucosylation product formed by beta-glucosidase action increased non-linearly, whereas that of glucose formed decreased non-linearly with increasing glycerol concentration. 6. All these results were found to fit the data calculated from rate equations derived on the basis of the proposed mechanism of enzyme action involving two ion-pair intermediates and a covalent alpha-D-glucosyl-enzyme in the reaction sequence [Umezurike (1987) Biochem. J. 241, 455-462].

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