The effect of pH on the transpeptidation and hydrolytic reactions of rat kidney γ-glutamyltransferase

Cook, Neil D.

doi:10.1016/0167-4838(85)90325-5

Cited by 19 publications

(19 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our results cannot exclude this possibility, which requires a change of rate-determining step at low pH from the acylation to the deacylation step. The pH dependence of the basic limb of the observed bell-shaped profile was attributed to the ionization of a kinetically critical acidic residue (54). Our pH-rate profile results confirm their observations and allow the assignment of a pK a value of 8.8 to the active site general acid.…”

Section: Scheme 2 γ-Glutamyl Transpeptidase Kinetic Studiessupporting

confidence: 85%

Nonlinear Free Energy Relationship in the General-Acid-Catalyzed Acylation of Rat Kidney γ-Glutamyl Transpeptidase by a Series of γ-Glutamyl Anilide Substrate Analogues

et al. 2001

View full text Add to dashboard Cite

The gamma-glutamyl transpeptidase (GGT) purified from rat kidney reacts with a series of eight parasubstituted L-glutamyl gamma-anilides, in the presence of Gly-Gly, catalyzing the formation of gamma-Glu-Gly-Gly (pH 8.0, 37 degrees C). The transpeptidation reaction was followed through the discontinuous colorimetric determination of the concentration of released parasubstituted aniline. Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each anilide substrate. A Hammett plot constructed by the correlation of log(k(cat)) and the sigma(-) parameter for each anilide substrate displays statistically significant upward curvature, consistent with a general-acid-catalyzed acylation mechanism in which the geometry of the transition state changes with the nature of the para substituent. Kinetic isotope effects were measured and are consistent with a reaction involving a proton in flight at the rate-limiting transition state. The pH-rate profiles measured over pH 7.0-9.5 are bell-shaped with kinetic pK(a) values that may be attributed to the active site nucleophile (or its general-base catalytic partner) and the active-site general acid. The variation of the latter pK(a) value as a function of temperature is consistent with an enthalpy of ionization expected for an ammonium ion acting as a general acid. Examination of the variation of k(cat) as a function of temperature gave values for the enthalpy and entropy of activation that are similar to those determined for the general-acid-catalyzed breakdown of the tetrahedral intermediate formed during acylation of chymotrypsin by similar amide substrates.

show abstract

Section: Scheme 2 γ-Glutamyl Transpeptidase Kinetic Studiessupporting

confidence: 85%

Nonlinear Free Energy Relationship in the General-Acid-Catalyzed Acylation of Rat Kidney γ-Glutamyl Transpeptidase by a Series of γ-Glutamyl Anilide Substrate Analogues

et al. 2001

View full text Add to dashboard Cite

show abstract

“…Mammalian c-GT showed, instead, bell-shaped curves for the transpeptidation reaction [43], whereas the hydrolase activity was usually lower and much less sensitive to pH changes [44]. Moreover, the transpeptidation reaction catalyzed by mammalian c-GT is up to 180-fold faster than hydrolysis [45], whereas our results indicate, for B. subtilis c-GT, a two-fold increase in transpeptidase activity with respect to hydrolysis at pH 10.…”

Section: Ph-activity Profilecontrasting

confidence: 57%

“…However, those results were obtained at a lower pH value (8.5), below the pK a of the glutamine amino group. It was proposed that an alkaline pH is beneficial for the c-GT-catalyzed transpeptidation reaction, in that it increases the concentration of unprotonated nucleophilic amino groups [44]. In our case, the autotranspeptidation reaction was mainly observed at pH 9.8, which is slightly above 9.1, the pK a of the amino group of glutamine.…”

Section: Ph-dependent Behavior Of B Subtilis C-gt Towards Glutaminementioning

confidence: 47%

“…Glycylglycine was early recognized as a very good acceptor for c-GT-catalyzed transpeptidation reactions [11]; it therefore became the substrate of choice and the reference compound for such studies. This was attributable to the low pK a (8.2) of its nucleophilic amino group with respect to those of other amino acids [44]. The low pK a of the glycylglycine amino group ensures that a large proportion of deprotonated, reactive nucleophilic species is present, even at those pH values at which the amino groups of standard amino acids are mainly in the protonated, unreactive form.…”

Section: Ph Dependence Of the Transpeptidation And Autotranspeptidatimentioning

confidence: 99%

See 1 more Smart Citation

pH‐Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ‐glutamyltransferase

et al. 2013

View full text Add to dashboard Cite

c-Glutamyltransferases (c-GTs) are heterodimeric enzymes that catalyze the transfer of a c-glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate c-glutamyl enzyme. In our search for a c-GT from a generally recognized as safe microorganism suitable for the production of c-glutamyl derivatives with flavor-enhancing properties intended for human use, we cloned and overexpressed the c-GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis c-GT in reactions involving glutamine as the donor compound and various acceptor amino acids. The common thread emerging from our results is a strong dependence of the hydrolase, transpeptidase and autotranspeptidase activities of B. subtilis c-GT on pH, also in relation to the pK a of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor molecule, undergoes rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to four c-glutamyl moieties are linked to a single glutamine. Moreover, we found that D-glutamine is also recognized both as a donor and as an acceptor substrate. Our results prove that the B. subtilis c-GT-catalyzed transpeptidation reaction is feasible, and the observed activities of c-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material c-polyglutamic acid.

show abstract

“…The optimum pH of the reaction medium for transpeptidation depends on the pK a of the amino group of the acceptor substrate, which should be in the unprotonated form in order to be competent as a nucleophile. 51,52 Although the transpeptidation-to-hydrolysis ratio for mammalian GGT is usually higher and better controlled through pH adjustment, 53 bacterial GGTs are preferred as biocatalysts for preparative purposes. They are easily produced and puried, 41,[54][55][56][57][58] avoiding the risk of viral contamination associated with an enzyme isolated from an animal organ.…”

Section: Introductionmentioning

confidence: 99%

Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant B. subtilis γ-glutamyltransferase (GGT)

et al. 2019

View full text Add to dashboard Cite

show abstract

The effect of pH on the transpeptidation and hydrolytic reactions of rat kidney γ-glutamyltransferase

Cited by 19 publications

References 12 publications

Nonlinear Free Energy Relationship in the General-Acid-Catalyzed Acylation of Rat Kidney γ-Glutamyl Transpeptidase by a Series of γ-Glutamyl Anilide Substrate Analogues

Nonlinear Free Energy Relationship in the General-Acid-Catalyzed Acylation of Rat Kidney γ-Glutamyl Transpeptidase by a Series of γ-Glutamyl Anilide Substrate Analogues

pH‐Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ‐glutamyltransferase

Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant B. subtilis γ-glutamyltransferase (GGT)

Contact Info

Product

Resources

About