Kinetics of in-out conformational change in PHBH 

 

In-Out Transition

H72N pH 6.5

16.2 s-1

 

 

 

H72N pH 10

14.8 s-1

 

 

 

WT pH 6.5

21.0 s-1

 

 

 

WT pH 10.0

33.6 s-1

 

 

 

WT+6HNA

75.8 s-1

 

 

 

Out-In Transition

H72N pH 6.5

27%

3.1 s-1

73%

18.0 s-1

H72N pH 10

43%

1.6 s-1

57%

15.8 s-1

WT pH 6.5

5.7 s-1

 

 

 

WT pH 10

14.0 s-1

 

 

 

WT+6HNA

5.9 s-1

 

 

 







 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Addition of a substrate analog (6-hydroxynicotinoic acid or 6HNA) significantly increased the rate of the in to out transition, the first step in the oxidation process. PHBH incubated with p-aminobenzoate, which is known to competitively inhibit the reaction of PHBH with its natural substrate, did not show fluctuations in fluorescence. This suggests the in-out conformational change may indeed serve as a control mechanism The mutant H72N, which lacks a proton transfer network  believed to be necessary for the in to out transition, shows a somewhat slowed transition rate.


Static and Dynamic Heterogeneity =>

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