Proton relay system in the active site of maltodextrinphosphorylase via hydrogen bonds with large proton polarizability: an FT-IR difference spectroscopy study.

Abstract

Maltodextrinphosphorylase (MDP) was studied in the pH range 5.4-8.4 by Fourier transform infrared (FT-IR) spectroscopy. The pK(a) value of the cofactor pyridoxalphosphate (PLP) was found between 6.5 and 7.0, which closely resembles the second pK(a) of free PLP.FT-IR difference spectra of the binary complex of MDP + alpha-D-glucose-1-methylenephosphonate (Glc-1-MeP) minus native MDP were taken at pH 6.9. Following binary complex formation, two Lys residues, tentatively assigned to the active site residues Lys533 and Lys539, became deprotonated, and PLP as well as a carboxyl group, most likely of Glu637, protonated. A system of hydrogen bonds which shows large proton polarizability due to collective proton tunneling was observed connecting Lys533, PLP, and Glc-1-MeP. A comparison with model systems shows, furthermore, that this hydrogen bonded chain is highly sensitive to local electrical fields and specific interactions, respectively. In the binary complex the proton limiting structure with by far the highest probability is the one in which Glc-1-MeP is singly protonated. In a second hydrogen bonded chain the proton of Lys539 is shifted to Glu637. In the binary complex the proton remains located at Glu637. In the ternary complex composed of phosphorylase, glucose-1-phosphate (Glc-1-P), and the nonreducing end of a polysaccharide chain (primer), a second proton may be shifted to the phosphate group of Glc-1-P. In the doubly protonated phosphate group the loss of mesomeric stabilization of the phosphate ester makes the C1-O1 bond of Glc-1-P susceptible to bond cleavage. The arising glucosyl carbonium ion will be a substrate for nucleophilic attack by the nonreducing terminal glucose residue of the polysaccharide chain.