Score: 1.00 |
|---|
| Title: Catalytic mechanism of scytalone dehydratase : site-directed mutagenisis , kinetic isotope effects , and alternate substrates .
| | Author: Basarab GS Steffens JJ Wawrzak Z Schwartz RS Lundqvist T Jordan DB .
| | Journal: Biochemistry Citation: V : 38 ( 19 ) P : 6012-24 Year: 1999 Type: ARTICLE | | Literature: oryza Field: abstract Doc ID: pub10320327 Accession (PMID): 10320327 | | Abstract: On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [ Lundqvist , T , Rice , J , Hodge , C N , Basarab , G S , Pierce , J and Lindqvist , Y ( 1994 ) Structure ( London ) 2 , 937-944 ] , eight active-site residues were mutated to examine their roles in the catalytic mechanism .
All but one residue ( Lys73 , a potential base in an anti elimination mechanism ) were found to be important to catalysis or substrate binding .
Steady-state kinetic parameters for the mutants support the native roles for the residues ( Asn131 , Asp31 , His85 , His110 , Ser129 , Tyr30 , and Tyr50 ) within a syn elimination mechanism .
Relative substrate specificities for the two physiological substrates , scytalone and veremelone , versus a Ser129 mutant help assign the orientation of the substrates within the active site .
His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination .
The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrates carbonyl through a water molecule .
Studies on a synthetic substrate , which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate ( vermelone ) , suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates .
All mutant enzymes bound a potent active-site inhibitor in near 1 : 1 stoichiometry , thereby supporting their active-site integrity .
An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained , likely accounting for its residual catalytic activity .
Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2 . 7 on kcat and 4 . 2 on kcat/Km , suggesting that steps after dehydration partially limit kcat .
Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat . kcat/Km with scytalone has a pKa of 7 . 9 similar to the pKa value for the ionization of the substrates C6 phenolic hydroxyl , whereas kcat was unaffected by pH , indicating that the anionic form of scytalone does not bind well to enzyme .
With an alternate substrate having a pKa above 11 , kcat/Km had a pKa of 9 . 3 likely due to the ionization of Tyr50 .
The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7 . 0 and 25 degrees C The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1 . 2 at pH 7 . 0 and 25 degrees C , and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate .
Thus , scytalone dehydrates through an E1cb mechanism off the enzyme .
| Matching Sentences:
[ Sen. 3, subscore: 1.00 ]: Steady-state kinetic parameters for the mutants support the native roles for the residues ( Asn131 , Asp31 , His85 , His110 , Ser129 , Tyr30 , and Tyr50 ) within a syn elimination mechanism .
| | Supplemental links/files: reference in endnote online text related articles pubmed citation | |
Score: 1.00 |
|---|
| Title: Structure-based design of potent inhibitors of scytalone dehydratase : displacement of a water molecule from the active site .
| | Author: Chen JM Xu SL Wawrzak Z Basarab GS Jordan DB .
| | Journal: Biochemistry Citation: V : 37 ( 51 ) P : 17735-44 Year: 1998 Type: ARTICLE | | Literature: oryza Field: abstract Doc ID: pub9922139 Accession (PMID): 9922139 | | Abstract: Scytalone dehydratase ( SD ) is a molecular target of inhibitor design efforts aimed at protecting rice plants from the fungal disease caused by Magnaporthe grisea .
As determined from X-ray diffraction data of an SD-inhibitor complex [ Lundqvist et al ( 1994 ) Structure ( London ) 2 , 937-944 ] , there is an extended hydrogen-bonding network between protein side chains , the inhibitor , and two bound water molecules .
From models of SD complexed to quinazoline and benztriazine inhibitors , a new class of potent SD inhibitors involving the displacement of an active-site water molecule were designed .
We were able to increase inhibitory potency by synthesizing compounds with a nitrile functionality displayed into the space occupied by one of the crystallographic water molecules .
Sixteen inhibitors are compared .
The net conversion of potent quinazoline and benztriazine inhibitors to cyanoquinolines and cyanocinnolines increased binding potency 2-20-fold .
Replacement of the nitrile with a hydrogen atom lowered binding affinity 100-30 , 000-fold .
X-ray crystallographic data at 1 . 65 A resolution on a SD-inhibitor complex confirmed that the nitrile functionality displaced the water molecule as intended and that a favorable orientation was created with tyrosines 30 and 50 which had been part of the hydrogen-bonding network with the water molecule .
Additional data on inhibitors presented herein reveals the importance of two hydrogen-bonding networks toward inhibitory potency : one between Asn131 and an appropriately positioned inhibitor heteroatom and one between a bound water molecule and a second inhibitor heteroatom .
| Matching Sentences:
[ Sen. 9, subscore: 1.00 ]: Additional data on inhibitors presented herein reveals the importance of two hydrogen-bonding networks toward inhibitory potency : one between Asn131 and an appropriately positioned inhibitor heteroatom and one between a bound water molecule and a second inhibitor heteroatom .
| | Supplemental links/files: reference in endnote online text related articles pubmed citation | |
