Phenolsulfophthalein dyes as probes in the study of lysozyme activity towards cell wall substrates.
Difference spectra have shown that the dissociation constant associated with the dominant species, formed by the binding of bromophenol blue or bromocresol purple to lysozyme, is not sensitive to pH in the range 6-9.5. This was confirmed from temperature-jump studies. However, the inhibition of lysozyme catalysed cell lysis by these dyes is dependent on pH and ionic strength. In the reaction scheme, which takes note of both these observations, we have to consider the formation of an enzyme-dye substrate complex (EDS) which has the same kcat as does the enzyme-substrate complex (ES). The formation of EDS from ES and free dye (D) is controlled by an ionisable group. Analysis of the data using an equation similar to that of Maurel and Douzou gives a pK of approx. 5.6 for this group and this pK is close to that of histidine-15. The inhibition mainly comes from the difference in the formation constants of EDS and ES. The initial binding site of the substrate (S) in ES is not in the cleft region A-F. The cell lysis takes place after S binds in the cleft, in a subsequent step. The rate constants of this step are included in kcat. (kcat is obtained by analysing activity using the simple Michaelis-Menten kinetics). Inhibition by chitotriose also supports this conclusion. The dye binding site is also suggested to be close to histidine-15. Experimental results support the contention that the electrostatic potential due to the negatively charged cell wall substrate could alter the effective pK of ionisable groups on the enzyme in ES.
D000818:Animals; D001962:Bromcresol Purple / Q000494:pharmacology*; D001978:Bromphenol Blue / Q000494:pharmacology*; D002645:Chickens; D003408:Cresols / Q000494:pharmacology*; D004529:Egg White; D005260:Female; D007700:Kinetics; D008433:Mathematics; D009113:Muramidase / Q000378:metabolism*; D010636:Phenols / Q000494:pharmacology*; D011485:Protein Binding; D013696:Temperature
D003408:Cresols; D010636:Phenols; D001978:Bromphenol Blue; D001962:Bromcresol Purple; D009113:Muramidase