Pyridoxal-5′-deoxymethylenephosphonate reconstituted D-serine dehydratase: A phosphorus-31 nuclear magnetic resonance study

Abstract

The phosphonate group of pyridoxal 5′-deoxymethylenephosphonate has a more favorable pK value than the phosphate group of pyridoxal-P to test the capabilities of this cofactor analogue to undergo specific ionic interactions with amino groups of model systems and amino acid side chains of D-serine dehydratase. Therefore, pyridoxal 5′-deoxymethylenephosphonate reconstituted, active D-serine dehydratase has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. The 31P chemical shift of the phosphonate group of the cofactor analogue is pH dependent with a pKa = 7.4, indicating exposure of the phosphonate group to solvent. Binding of the competitive inhibitor isoserine results in the formation of the isoserine-cofactor analogue complex. This transaldimination complex is fixed to the enzyme via a salt bridge of the dianionic phosphonate group of the cofactor analogue indicated by an apparent pK shift of the phosphonate group towards more acidic pH values. Similar shifts of the apparent pK value were observed for the corresponding Schiff base with n-dodecylamine when placed into a micellular environment. Addition of hexadecyltrimethylammonium bromide generating mixed micelles leads to intermediary pK values.