Chemistry Reference
In-Depth Information
of a DNA substrate, the number and location of metal ions vary among MutH-like
type IIP REases,
65
e.g. no metal ion in the Eco RI-substrate complex
72
and three
possible binding sites in Eco RV.
73
Accompanying the metal ion variations, the active
site composition of type IIP REases differs in the number of carboxylates required
(two, three or four), and Lys of the DEK motif, catalytically essential for most of
MutH-like endonucleases, is replaced by Glu in BamHI and Gln in BglII.
67
These
observations have raised speculation that REases may have different catalytic
mechanisms using varying numbers of divalent cations.
65,74
The crystal structures of a bacterial RNase H complexed with RNA/DNA
hybrid substrates show two Mg
2+
ions bound in the active site at a magnesium con-
centration below 2.5 mM.
63
In the absence of a substrate, two Mn
2+
ions were found
at similar positions, but only at metal-ion concentrations much higher than are
physiologically relevant.
68
In the enzyme-substrate complexes, the two metal ions
are jointly coordinated by the conserved Asp and scissile phosphate, each of which
functions as a bidentate chelate (Figure 15.4A). By convention, the metal ion lig-
anded to the nucleophile is called A, and the one liganded to the leaving group is
B. A similar substrate-dependent two-metal-ion coordination is observed in crystal
structures of the RNase H-like Tn5 transposase-DNA complexes.
64
Among MutH-like nucleases, in the presence of their respective cognate sub-
strates, MutH, BamHI and BglI bind two metal ions in the active site, in spite of the
catalytic residue differences with Lys in MutH and BglI and Glu in BamHI.
67
The
coordination of the two metal ions by the scissile phosphate and a conserved Asp
resembles the metal coordination observed in polymerases and RNase H (Figures
15.3 and 15.4), which is hereby referred to as canonical. It appears that in all cases
at least one Asp is needed for coordination of the two metal ions. Asp is probably
preferred because it has fewer rotamer conformations than Glu and costs less
entropy to be fi xed in one conformation. The metal ions observed in type IIP
REases, PvuII, EcoRI and EcoRV, however, differ from the canonical ones in
number and location.
73,75,76
Such differences were originally attributed to different
compositions of catalytic residues and perhaps different catalytic mechanisms. The
conserved active site residues and tertiary structures of MutH, EcoRI PvuII and
EcoRV suggest that these enzymes most probably share the same catalytic mecha-
nism. The question is how to reconcile the metal ion differences observed in these
related enzyme-substrate complexes.
15.3.1 Binding of Two Metal Ions in the Active Site Is Substrate Dependent
Recently, crystal structures of human RNase H complexes with a RNA/DNA hybrid
have provided new evidence on the substrate dependence of metal ion binding.
77
One of the crystal forms contains 12 protein molecules in one asymmetric unit.
Depending on the crystal lattice contacts, in some protein molecules the scissile
phosphate is shifted away from the active residues by 1-2 Å, and these molecules
contain only a single instead of two Ca
2+
(Mg
2+
mimics), and the Ca
2+
binding site
is neither the canonical A or B. This shows that not only are the number and loca-
tion of divalent cations in the active site dependent on the presence of substrate,
