Anhydride Intermediates in Catalysis by Pepsin: Is Pepsin an Enzyme with Two Active Sites?

Kaiser, E. T.; Nakagawa, Yasushi

doi:10.1007/978-1-4757-0719-9_10

Cited by 3 publications

(1 citation statement)

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“…The material that accumulates during this lag is (a) like pepsin but unlike pepsinogen in being pH 8.5 labile but is (b) unlike pepsin and like pepsinogen in having the activation peptide attached to it. It is possible that this is a covalent intermediate formed by transpeptidation, analogous to the acyl-enzyme proposed for pepsin catalysis (Fruton, 1976;Kaiser & Nakagawa, 1977), or it may be the noncovalent complex of 0-pepsin and peptide that may accumulate at higher pH values.2 Concerning these early events in pepsinogen activation, we have evidence for a change in fluorescence of pepsinogen that is complete 50 ms following acidification, but we do not yet have quantitative data to report.…”

Section: Discussionmentioning

confidence: 89%

Preparation and activation of a spin-labeled pepsinogen

Twining¹,

Sealy²,

Glick³

1981

Biochemistry

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gen derivative has been prepared, using 3-[[2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrolyl-1-oxy with 3 spin-labels/protein molecule. Two are located in the peptide that is released first (1-16) and the other is in the secondarily removed sequence (17-44). Activation of the labeled zymogen is a faithful model of native pepsinogen activation because the two processes are closely related in rate, pH dependence of rate, and sites of peptide bond cleavage. The ESR signal associated with the bound label changes during activation due to release of the activation peptide. The rate of release (0.5 min(-1) at pH 2, 22 degrees C) is an order of magnitude slower than is the rate of activation, i.e., cleavage of the peptide bond that holds the peptide to the enzyme. Activation in the presence of pepstatin, however, results in peptide release (2 min(-1) at pH 2, 22 degrees C) nearly as fast as activation occurs. At pH values between 2.5 and 3, there is a lag in the change of the ESR signal following acidification. This indicates accumulation of an intermediate with pH 8.5 labile activity that still has its activation peptide attached. The strong temperature dependence of the rate of activation (26 kcal/mol) is reflected neither in reported characteristics of pepsin catalysis nor in the measured rate of release of the activation peptide from the enzyme (13 kcal/mol).

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Section: Discussionmentioning

confidence: 89%