scholarly journals The necessity of magnesium cation for acid assistance aglycone departure in catalysis by Escherichia coli (lacZ) β-galactosidase

1978 ◽  
Vol 175 (2) ◽  
pp. 539-546 ◽  
Author(s):  
M L Sinnott ◽  
S G Withers
Keyword(s):  
Escherichia Coli ◽  
Isotope Effects ◽  
Free Enzyme ◽  
Pyridinium Salts ◽  
Kinetic Isotope ◽  
Rate Limiting Step ◽  
Magnesium Cation ◽  
Rate Of Hydrolysis ◽  
Rate Limiting ◽  
Hydrolysis Of

1. Removal of Mg2+ from Escherichia coli (lacZ) beta-galactosidase slightly increases the rate of hydrolysis of galactosyl pyridinium salts, but decreases the rate of hydrolysis of arylgalactosides. 2. Fair correlation of logkcat. and log (Km) with the pKa of aglycone is now observed for arglygalactosides, as well as for glycosyl pyridinium salts. 3. Degalactosylation of Mg2+-free enzyme is the rate-limiting step in the hydrolysis of 2,4-dinitrophenyl galactoside. 4. alpha-Deuterium kinetic isotope effects for both sets of substrates are consistent with the rate-determining generation of a glycosyl cation. 5. The pH-independent, SNl hydrolysis of 3,4-dinitrophenyl galactoside has been measured: it is as fast as that of the galactosyl 3-chloropyridinium ion. 6. Hydrolysis of these two substrates by Mg2+-free enzyme proceeds at very similar rates. 7. It is concluded that loss of both types of aglycone takes place, without acid catalysis, from the first ES complex of substrate and apoenzyme. 8. Data for galactosyl azide and thiopicrate confirm that neither charge nor change of atom is the cause of the differences in behavior between aryl galactosides and galactosylpyridinium salts.

scholarly journals The β-galactosidase-catalysed hydrolyses of β-d-galactopyranosyl pyridinium salts. Rate-limiting generation of an enzyme-bound galactopyranosyl cation in a process dependent only on aglycone acidity

1974 ◽  
Vol 143 (3) ◽  
pp. 751-762 ◽  
Author(s):  
Michael L. Sinnott ◽  
Stephen G. Withers
Keyword(s):  
Acid Catalysis ◽  
Isotope Effects ◽  
Pyridinium Salt ◽  
Pyridinium Salts ◽  
Kinetic Isotope ◽  
Rate Limiting Step ◽  
Aryl Glycosides ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Derivatives Of

1. β-d-Galactopyranosyl pyridinium salts are well-behaved substrates for the β-galactosidase of Escherichia coli, catalysis occurring by the interaction of the salt itself with the normal active site of the protein. 2. logkcat. values for seven such salts show a linear relationship (correlation coefficient=−0.997) with the pKa of the parent pyridine. 3. The β-d-galactopyranosyl derivatives of pyridine and 4-bromoisoquinoline exhibit α-deuterium kinetic isotope effects of 1.136±0.040 and 1.187±0.046 on their enzymic hydrolysis, indicating formation of a galactopyranosyl cation in the rate-limiting step. 4. This behaviour of the pyridinium salts contrasts with the behaviour of aryl galactosides and this contrast can be accommodated by the β-galactosidase mechanism of Sinnott & Souchard (1973). 5. The α-deuterium kinetic isotope effect for the hydrolysis of β-d-galactopyranosyl azide is 1.098±0.033; comparison of the kcat. value of the azide with that of a pyridinium salt of the same aglycone pKa enables a maximum factor of 70 to be ascribed to the acceleration of the departure of azide by intracomplex general acid catalysis. 6. The possibility of the rate-limiting process in the glycosidase-catalysed hydrolysis of aryl glycosides being a conformation change is considered for a number of glycosidases where correlations of kcat. with aglycone acidity, reported in the literature, have been unsuccessful.

Growth and cellulolytic activity of Cellulomonas flavigena

1981 ◽  
Vol 27 (12) ◽  
pp. 1260-1266 ◽  
Author(s):  
B. H. Kim ◽  
J. W. T. Wimpenny
Keyword(s):  
Free Enzyme ◽  
Crystalline Cellulose ◽  
Exponential Growth Phase ◽  
Cellulolytic Bacterium ◽  
Cellulolytic Activity ◽  
Mineral Salts ◽  
Cellulomonas Flavigena ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Hydrolysis Of

Growth factor requirements, growth kinetics, and the ability to produce the enzyme cellulase were examined in the cellulolytic bacterium Cellulomonas flavigena KIST 321. The organism was found to require only thiamine for growth in mineral salts medium containing simple sugars or cellulose. Growth rates on various carbohydrates suggested that disruption of the crystalline structure was the rate-limiting step in the utilization of crystalline cellulose, and hydrolysis of the polymer itself was as rapid as the uptake of the hydrolytic product. When the organism was grown on cellulose the cellulolytic activity appeared to be bound to the cell at the beginning of the exponential growth phase: only after this did cell-free enzyme activity appear. The cell-free enzyme appeared to be unstable, and its activity decreased at the beginning of the stationary phase.

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 Inverse Solvent Isotope Effects in Enzyme-Catalyzed Reactions

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1933
Author(s):  
Patrick L. Fernandez ◽  
Andrew S. Murkin
Keyword(s):  
Isotope Effect ◽  
Isotope Effects ◽  
Solvent Isotope Effect ◽  
Kinetic Isotope ◽  
Rate Limiting Step ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Solvent Isotope ◽  
Rate Limiting ◽  
Solvent Isotope Effects

Solvent isotope effects have long been used as a mechanistic tool for determining enzyme mechanisms. Most commonly, macroscopic rate constants such as kcat and kcat/Km are found to decrease when the reaction is performed in D2O for a variety of reasons including the transfer of protons. Under certain circumstances, these constants are found to increase, in what is termed an inverse solvent kinetic isotope effect (SKIE), which can be a diagnostic mechanistic feature. Generally, these phenomena can be attributed to an inverse solvent equilibrium isotope effect on a rapid equilibrium preceding the rate-limiting step(s). This review surveys inverse SKIEs in enzyme-catalyzed reactions by assessing their underlying origins in common mechanistic themes. Case studies for each category are presented, and the mechanistic implications are put into context. It is hoped that readers may find the illustrative examples valuable in planning and interpreting solvent isotope effect experiments.

Kinetic Isotope Effects on the Rate-Limiting Step of Heme Oxygenase Catalysis Indicate Concerted Proton Transfer/Heme Hydroxylation

2003 ◽  
Vol 125 (52) ◽  
pp. 16208-16209 ◽  
Author(s):  
Roman Davydov ◽  
Toshitaka Matsui ◽  
Hiroshi Fujii ◽  
Masao Ikeda-Saito ◽  
Brian M. Hoffman
Keyword(s):  
Proton Transfer ◽  
Heme Oxygenase ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

scholarly journals A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) β-galactosidase

1990 ◽  
Vol 268 (2) ◽  
pp. 317-323 ◽  
Author(s):  
T Selwood ◽  
M L Sinnott
Keyword(s):  
Escherichia Coli ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Effect Study ◽  
Solvent Isotope Effect ◽  
Kinetic Isotope ◽  
Solvent Isotope ◽  
Paper Abstract ◽  
Hydrolysis Of

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl β-D-galactopyranoside and 3,4-dinitrophenyl β-D-galactopyranoside Escherichia coli (lacZ) β-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl β-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl β-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl β-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl β-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the ‘conformation change’ in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

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