Kinetics of Iterative Carbohydrate Transfer to Polysaccharide Catalyzed by Chondroitin Polymerase on a Highly Sensitive Flow‐Type 27 MHz Quartz‐Crystal Microbalance

Abstract

Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 μM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.

Sweet but Challenging: Tackling the Complexity of GAGs with Engineered Tailor‐Made Biomaterials

“…In the past, hydrazone ligation was a popular method for conjugating the reducing end of GAGs. [287][288][289] However, it was found to be inefficient for long GAG chains and unstable in aqueous solution. [290] Functionalization of GAG reducing end through oxime ligation is now favored, as it shows both improved yields and stability.…”

Single-Molecular Enzymatic Elongation of Hyaluronan Polymers Visualized by High-Speed Atomic Force Microscopy

“…10,11 We elucidated the mechanisms of glycosyltransferases by determining the kinetic parameters for enzymatic elongation of a chondroitin polymer, using a flow-type high-sensitivity QCM. 12 As another example, hyaluronan, is an important glycosaminoglycan involved as a biological lubricant and in phylaxis of bacteria. However, kinetic studies of hyaluronan synthases are still vague, and k cat values varying from 1 to 100 s −1 have been reported.…”

“…We have used a quartz crystal microbalance (QCM) technique to follow the enzyme–substrate complex intermediate of the enzyme reaction as the mass changes and obtained kinetic parameters for both enzyme-binding and catalytic processes for glycosidase and glycosyltransferase . Glycosaminoglycans play an important role as extracellular matrices in living systems, and there are many unsolved questions concerning their production processes and the mechanisms of the related emzymes. , We elucidated the mechanisms of glycosyltransferases by determining the kinetic parameters for enzymatic elongation of a chondroitin polymer, using a flow-type high-sensitivity QCM . As another example, hyaluronan, is an important glycosaminoglycan involved as a biological lubricant and in phylaxis of bacteria.…”