alpha-Chymotrypsin: Enzyme concentration and kinetics

Bender, Myron L.; Kézdy, Ferenc J.; Wedler, F. C.

doi:10.1021/ed044p84

Cited by 190 publications

(138 citation statements)

References 11 publications

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“…3e; k3). This "ping-pong" mechanism is well established for -chymotrypsin, which displays similar profiles with pNP esters and proceeds via an ester intermediate; 42 and it is similar to that proposed for previously designed hydrolase catalysts. 19 Rapid and stoichiometric acylation within the heptameric barrel leads to seven acylated Cys side chains, followed by rate-limiting hydrolysis of the thioester intermediate.…”

Section: Discussionsupporting

confidence: 79%

“…This has been used for the reaction of -chymotrypsin with pNP esters to deliver k2/Ks and kcat/KM, where KS is the pseudo KM for the acylation step. 42 In our case, this model did not fit the experimental data (SI Fig. 1.11-1).…”

Section: Cc-hept-c-h-e Has Enzyme-like Behaviourmentioning

confidence: 56%

“…3a), reminiscent of a "ping-pong" mechanism involving a covalently bound acyl intermediate as observed for the serine protease -chymotrypsin with similar substrates. 42 Furthermore, the y-intercept for the second, steady state curve of this profile matched the concentration of peptide in the assay.…”

Section: Cc-hept-c-h-e Has Enzyme-like Behaviourmentioning

confidence: 85%

“…Data were background corrected (measured in the presence of TCEP), and errors obtained from the standard deviation of the repeats. Fitting of the obtained curves was carried out following the method of Bender et al 42 Where k2, KS, kcat and KM can be obtained by fitting of pre-steady state kinetic curves to an equation with an exponential and a linear term. In the case of CC-Hept-C-H-E and CC-Hept-hC-H-E fitting to a double exponential and a linear term returned a good fit to the data (SI Fig.…”

Section: Kinetic Assaysmentioning

confidence: 99%

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Installing hydrolytic activity into a completely de novo protein framework

et al. 2016

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Designing enzyme-like catalysts tests our understanding of sequence-tostructure/function relationships in proteins. Here, we install hydrolytic activity predictably into a completely de novo and thermo-stable -helical barrel, which comprises 7 helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses pnitrophenyl acetate with catalytic efficiencies matching the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.(134 words)

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Section: Discussionsupporting

confidence: 79%

Section: Cc-hept-c-h-e Has Enzyme-like Behaviourmentioning

confidence: 56%

Section: Cc-hept-c-h-e Has Enzyme-like Behaviourmentioning

confidence: 85%

Section: Kinetic Assaysmentioning

confidence: 99%

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Installing hydrolytic activity into a completely de novo protein framework

et al. 2016

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“…Kinetic measurements were similar to those described previously 12-41. The k3 (ms) value, equal to kcat, for nitrophenyl esters [6,7] , Was determined under conditions of stationary cuchymotryptic hydrolysis of rrans-NPNC, with [S10 9 Km(app) (where Km (aPPJ < lo-' M). The rate of pnitrophenolate ion liberation was followed spectrophotometrically (400 nm)at [S],, = 1.7 X lo-' M; [El0 was varied from 7X 10-8Mto2X lo-'M.…”

Section: Methodsmentioning

confidence: 99%