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UID:1051@biotech.technion.ac.il
DTSTART;TZID=Asia/Jerusalem:20210721T170000
DTEND;TZID=Asia/Jerusalem:20220202T164952
DTSTAMP:20220512T124718Z
URL:https://biotech.technion.ac.il/events/allosteric-activation-of-the-b-s
 ubtilis-aminopeptidase-bsap-towards-a-perfect-enzyme-2/
SUMMARY:Allosteric activation of the B. subtilis aminopeptidase BSAP: towar
 ds a “perfect” enzyme
DESCRIPTION:Aminopeptidases catalyze the cleavage of a single amino acid fr
 om the amino terminus of peptides and proteins and are involved in many cr
 ucial biological processes. The goal of the current study was to explore t
 he structure-function relationship of the bacterial metallo-aminopeptidase
  from Bacillus subtilis (BSAP). The detailed three-dimensional structure o
 f BSAP was determined by X-ray crystallography at 1.7Å resolution. The 46
 kDa enzyme consists of two main domains\; a TIM barrel catalytic domain (A
 A 32-100\, 226-455) containing two Zn molecules and a protease-associated 
 (PA) domain (AA 101-225). Interestingly\, the 23-AA C-terminus tail appear
 ed unstructured in the crystal structure\, with no clear electron density 
 for most of it. However\, Glu452 at the end of the tail interacted strongl
 y with one of the Zn ions inside the active site\, potentially interfering
  with substrate binding. To study the exact roles of the carboxy-terminus 
 tail of BSAP\, we prepared a series of truncated forms of the enzyme\, inc
 luding deletions and key amino acid replacements. The removal of the whole
  C-terminus tail did not affect the activity towards the chromogenic subst
 rate p-nitroanilide-Lys and several peptides examined\, suggesting that Gl
 u452 is not inhibiting activity. Unexpectedly\, however\, a single replace
 ment Glu452Asn resulted in a 3-orders of magnitude increase of kcat withou
 t affecting significantly KM\, providing a 3000-fold improvement in the sp
 ecificity constant kcat/KM. The δ-amine group of Asn452 appears to stabil
 ize the transition state\, thus reducing the free activation energy by up 
 to 6.6 Kcal/mole. Kinetic measurements at different viscosities indicated 
 the rate of hydrolysis is under diffusional-control\, consistent with the 
 observed value of the specificity constant\, 9.4x108 M-1s-1 against the he
 xa-peptide KAAAAW. Our results demonstrate that BASP can be activated allo
 sterically making it a “perfect enzyme”.
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