Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

104 Chapter 5
Figure 5.29 Structure of alkaline phosphatase (PDB Wle 1B8J), in which the catalytic Ser-102 has
been derivatised with vanadate, representing the phospho–serine intermediate. ModiWed Ser-102
and Arg-166 are highlighted in red. The two active site zinc ions are shown in black.
Protein tyrosine phosphatases, which catalyse the dephosphorylation of
phosphotyrosine peptides, also contain an active site arginine residue which
provides transition state stabilisation for phosphoryl transfer. 15 N kinetic isotope
eVects measured for these enzymes are consistent with extensive bond
cleavage to the leaving group in the transition state, consistent with a dissociative
transition state (mechanism A, Figure 5.28). Therefore, the active site
arginine residue appears to stabilise the metaphosphate intermediate ‘in Xight’.
Enzymes that cleave the phosphodiester backbone of RNA and DNA are
known as nucleases. Some nucleases are relatively non-speciWc, for example an
3 0 ! 5 0 exonuclease enzyme found in snake venom which will digest singlestranded
DNA from the 3 0 end successively. However, the endonuclease
enzymes usually have highly speciWc cleavage sites, for example the restriction
endonuclease EcoR1 (produced by Escherichia coli) cleaves at a speciWc six-base
sequence 5 0 -GAATTC-3 0 , making cuts on both strands of the DNA as shown in
Figure 5.31.
The best characterised nuclease enzyme is ribonuclease A, a 14-kDa
protein which was the Wrst protein to be reversibly unfolded and refolded
in the absence of other proteins. This enzyme cleaves RNA at
sites immediately following pyrimidine bases (cytidine or uridine), leaving a