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

Maltodextrinphosphorylase (MDP) was studied in the pH range 5.4-8.4 by Four ier transform infrared (FT-IR) spectroscopy. The pK(a) value of the cofacto r pyridoxalphosphate (PLP) was found between 6.5 and 7.0, which closely res embles the second py, of free PLP. FTIR difference spectra of the binary co mplex of MDP + alpha-D-glucose-1-methylenephosphonate (Glc-1-MeP) minus nat ive MDP were taken at pH 6.9. Following binary complex formation. two Lys r esidues, 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 pol arizability due to collective proton tunneling was observed connecting Lys5 33, PLP, and Glc-1-MeP. A comparison with model systems shows, furthermore. that this hydrogen bonded chain is highly sensitive to local electrical fi elds and specific interactions, respectively. In the binary complex the pro ton limiting structure with by far the highest probability is the one in wh ich Glc-1-MeP is singly protonated. Glc-1-MeP is singly protonated. In a se cond hydrogen bonded chain the proton of Lys539 is shifted to Glu637. in th e binary complex the proton remains located at Glu637. In the ternary compl ex composed of phosphorylase. glucose-1-phosphate (Glc-1-P), and the nonred ucing end of a polysaccharide chain (primer), a second proton may be shifte d to the phosphate group of Glc-1-P. In the doubly protonated phosphate gro up the loss of mesomeric stabilization of the phosphate ester makes the C-1 -O-1 bond of Glc-1-P susceptible to bond cleavage. The arising glucosyl car bonium ion will be a substrate for nucleophilic attack by the nonreducing t erminal glucose residue of the polysaccharide chain.