J. Biol. Chem., Vol. 263, Issue 17, 8011-8016, 06, 1988
Transpeptidation reactions of porcine pepsin. Formation of tetrapeptides from dipeptide substrates
MK Lutek, T Hofmann and CM Deber
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Pepsin-catalyzed transpeptidation was studied by high resolution 75 MHz 13C
nuclear magnetic resonance spectroscopy. Enrichment with 13C at the
carbonyl carbons of the substrates Leu-Tyr-NH2 and Leu-Leu-NH2 facilitated
detection and identification of the transpeptidation and hydrolysis
products of enzymic action. Porcine pepsin was found in each case to
synthesize and release the tetrapeptide Leu-Leu-Leu-Leu as the primary
product of transpeptidation, the longest oligomeric product of
transpeptidation observed to date. Productive binding of the dipeptide
substrates into the active site groove of pepsin required an induction
period of several minutes. Quenching experiments suggested the presence of
strongly bound intermediate forms of Leu and Leu-Leu prior to observation
of any enzyme-free products. The finding of the tetrapeptide as a primary
product is discussed as an instance where transpeptidation of the
tripeptide competes successfully with the action of pepsin subsite S3 as a
trigger for product release.
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