Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior

Table 1

Three variations of the Pendulum hypothesis.

Variant	Disaccharide assembly	Glycosyl-UDP sites
1	Sequential	Four independent sites
2	Simultaneous	Three independent sites
3	Alternating	Two or three dependent sites

The mechanism for adding sugars to the reducing end of HA could entail polymerization of a disaccharide unit by either a sequential (i.e., one sugar at a time) or a concerted (i.e., simultaneous) mechanism. For the sequential assembly of a disaccharide unit (variant 1), the enzyme would need two glycosyl-UDP binding sites for addition of each sugar, one for a HA-UDP and one for a sugar-UDP. Since there are two types of HA-UDP species, the enzyme would need four glycosyl-UDP binding sites to assemble each disaccharide unit. For disaccharide assembly at the reducing end in a concerted way (variant 2), HAS would require three glycosyl-UDP binding sites, one each for GlcNAc-UDP, GlcUA-UDP, and a specific HA-UDP donor chain (i.e., HA-GlcUA-UDP or HA-GlcNAc-UDP), depending on which of the two possible HA disaccharide units was assembled. Another variation is that HAS contains only one donor and one acceptor glycosyl-UDP binding site, whose specificities alternate as the two sugars are assembled one at a time (variant 3). If there is a single donor binding site, its specificity would alternately recognize HA-GlcUA-UDP and HA-GlcNAc-UDP. There could be two separate sugar-UDP sites, but if there is a single acceptor binding site, its specificity would also alternate in a reciprocal fashion with the HA-UDP site to bind GlcUA-UDP or GlcNAc-UDP.