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SUMMARY:X-ray crystallographic studies and quantum mechanical modeling of 
 the amidase reaction mechanism
DTSTART;VALUE=DATE-TIME:20110714T114500Z
DTEND;VALUE=DATE-TIME:20110714T120000Z
DTSTAMP;VALUE=DATE-TIME:20260911T105103Z
UID:indico-contribution-5111@events.saip.org.za
DESCRIPTION:Speakers: Bryan Trevor Sewell (University of Cape Town)\nThe a
 midases catalyse the conversion of amides to the corresponding carboxylic 
 acid and ammonia. We have visualized the structure of the amidase (MW= 30 
 kDa) from a bacterium\, Nesterenkonia sp.  at atomic resolution by X-ray c
 rystallography. The primary components of the active site are two negative
 ly charged carboxylates (E1 and E2) arising from two conserved glutamate s
 ide chains\, a positively charged amino group (K) arising from a conserved
  lysine\, a sulphydryl arising from a conserved cysteine and a water molec
 ule. Mutation of the active site residues has enabled us to visualize the 
 unreacted substrate and a number of artificial intermediate states which h
 ave led to our insights. We propose a reaction mechanism which passes thro
 ugh a thioester adduct to the cysteine.  Although this in itself is not co
 ntroversial\, our studies demonstrate for the first time how exquisitely a
 ccurate geometrical placement of the various groups leads to accurate ster
 eoelectronic alignment which allows the transition states to form. The con
 figuration of E1\, E2 and K ensures that K remains positively charged thro
 ughout the reaction thus enabling it to act as an acid catalyst and provid
 e an oxyanion hole to stabilize the tetrahedral transition states. The con
 figuration also accurately positions the substrate for nucleophilic attack
  by the sulphydryl by hydrogen bonding to the amide\, positions  E1 as a b
 ase catalyst and positions the water with the correct stereoelectronic ali
 gnment for a nucleophilic attack on the thioester. The transition state en
 ergies\, electron densities and hydrogen placements calculated using Gauss
 ian09 demonstrate the plausibility of our proposal.\n\nhttps://events.saip
 .org.za/event/7/contributions/5111/
LOCATION: Acro3
URL:https://events.saip.org.za/event/7/contributions/5111/
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