Sucrose phosphorylase inhibition

Extracts of Neisseria perflava contain phosphorylase also, but this could be differentiated from amylosucrase, which is specific for sucrose and has no action on a-D-glucosyl phosphate. The polysaccharide has been investigated by methylation and enzymic analysis, and the presence of branched chains of (1— 4)-linked a-D-glucose residues rigidly established. The acceptor specificity of amylosucrase has not been studied in detail, but maltosaccharides and small amylose-type molecules are logical acceptors, especially as amylosucrase action on sucrose is inhibited by a-amylase. 

It was at this state of knowledge that an incidental observation with extracts of C. kluy-veri led to a development which played a significant role in elucidating the mechanism of CoA action. It was observed that arsenate inhibited the ability of extracts to catalyze the oxidation of butyrate, as well as the reduction of acetyl-P and acetate to butyrate. The inhibition was ultimately explained by the fact that arsenate stimulated the enzymic hydrolysis of acetyl-P [reaction (18)]. By analogy to the postulated role of arsenate in the hydrolysis of glucose-l-P by sucrose phosphorylase,< > it was proposed that the arsenolysis of acetyl-P could be the result of a reversible transfer of the acetyl moiety to an acetyl acceptor, x , catalyzed by a phosphotransacetylase [reaction (21)]. 

One possible way to solve this problem is to combine another enzyme, like SP, which produces a-GlP for GP. Waldmann and colleagues reported the combined use of SP and GP for the production of amylose from sucrose (1986). In this system, SP catalyzes the phosphorolysis of sucrose to produce a-GlP and fructose, and the a-GlP is next used as a substrate of GP to produce amylose. An interesting feature of this SP-GP system is that Pi produced in the second GP reaction is used as a substrate for the first SP reaction. The cooperative action by the two phosphorylases proceeds continuously with a constant Pi concentration, without any inhibition caused by an accumulation of Pi. Based on this SP-GP system, we have now established the process to manufacture essentially linear amylose with controlled molecular size, by using thermostable variants of SP and GP (Yanase et al., 2007 Ohdan et al., 2007). 

When cultures or enzyme preparations of N. perfiava are allowed to act on a-D-glucose-l-phosphate, some amylopolysaccharide is produced, indicating the presence of a phosphorylase. However, the amylosucrase can be distinguished from the bacterial phosphorylase by its stability to heat and by the fact that the synthesis of the polysaccharide from sucrose is not suppressed in high concentration of inorganic phosphate. The synthetic reaction proceeds without a noticeable lag period, suggesting that primer material, if needed, is present in the preparations. Although there is no direct evidence for this, the fact that the formation of polysaccharide is completely inhibited in the presence of traces of salivary amylase indicates that a primer may be required. 

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