Question

With the aid of a simple generic diagram, identify and explain how the type(s) of chemical bonding stabilizes a secondary structure that is present in crystal structure of Yeast Hexokinase PII with the correct amino acid sequence

Answer 1

Hexokinase is the first enzyme in the glycolytic pathway, catalyzing the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII.

The crystal structure of yeast hexokinase PII from Saccharomyces cerevisiae without substrate or competitive inhibitor is determined and refined in a tetragonal crystal form at 2.2-A resolution. The folding of the peptide chain is very similar to that of Schistosoma mansoni and previous yeast hexokinase models despite only 30% sequence identity between them. Distinct differences in conformation are found that account for the absence of glucose in the binding site.

Comparison of the current model with S. mansoni and yeast hexokinase PI structures both complexed with glucose shows in atomic detail the rigid body domain closure and specific loop movements as glucose binds. A hydrophobic channel formed by strictly conserved hydrophobic residues in the small domain of the hexokinase is identified. The channel's mouth is close to the active site and passes through the small domain to its surface. The possible role of the observed channel in proton transfer is discussed.

A secondary, PTS-independent system for utilization of glucose also exists, consisting of glucose uptake by galactose permease, followed by phosphorylation by glucokinase to yield the metabolic intermediate glucose-6-phosphate. Although glk mutant strains of E. coli and Bacillus subtilis are not visibly physiologically impaired, this enzyme retains the important function of phosphorylating any free intracellular glucose.

All these secondary structure elements match those present in the human HK structure apart from three additional 310 helices found in the yeast HK structure. The structure of S. mansoni HK shows the same secondary structural pattern. Direct comparison with the previous HK structures [17-20] can only be somewhat approximate due to the limited refinement and the uncertain amino acid composition of those earlier models. However, such an approximate comparison suggests that most of the secondary structural elements in the current structure have their equivalents in the previous ones.

This clearly demonstrates extensive similarity between the N- and Cterminal halves of type I human hexokinase, rat hexokinase and hexokinase from S. mansoni, and between these and yeast hexokinase, consistent with the gene duplication-fusion concept proposed by Colowick. Approximately 34% of the amino acid residues are conserved in all members of the family, and 13% are perfect matches. The strong conservation of these residues implies their relevance to biological function. Various strictly conserved amino acid residues are present in the binding site. A high number of glycine residues are also conserved . These residues are located at the ends of β-strands or α-helices, changing the direction of the chain. Their conservation probably gives the hexokinase molecule the flexibility necessary for binding glucose and ATP.

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