Chemistry Reference
In-Depth Information
FIGURE 12.14
The zinc-binding sites of subclass B1 (BCII from B cereus), B2 (CphA from A. hydrophila), and B3 (FEZ1 L. gormanii)
b
-lactamases.
(From
Bebrone, 2007
. Copyright 2007 with permission from Elsevier.)
Zn
2
þ
ion which has the characteristics of a catalytic zinc site, while the role, and even the essentiality of the second
zinc atom, is not clear.
Aminopeptidases are counterparts to carboxypeptidases, removing N-terminal amino acids. However, unlike
the carboxypeptidases, they contain dinuclear zinc sites. They fall into two groups, the first of which includes the
leucine aminopeptidase from bovine lens, while the second includes the leucine aminopeptidases AAP from
Aeromonas proteolytica and SAP from Streptomyces griseus (
Figure 12.15
)
. The mechanism of the AAP enzyme
has been well studied, and may well represent a general catalytic mechanism for peptide hydrolysis by metal-
lopeptidases with a cocatalytic active site.ki.
The proposed general mechanism is represented in
Figure 12.16
. After the binding of the carbonyl oxygen
atom of the incoming substrate to Zn
1
, which polarizes the carbonyl group, rendering it susceptible to nucle-
ophilic attack, the bridging water/hydroxide becomes terminal and is coordinated to Zn
1
. The breaking of the
Zn
2
-OH(H) bond is probably assisted by N-terminal amine binding in aminopeptidases and C-terminal
carboxylate binding in carboxypeptidases to Zn
2
whose role is simply to position the substrate correctly in the
active site. Next, a glutamic acid residue (or a histidine) located near the catalytic active site assists in the
deprotonation of the terminal water molecule, giving a nucleophilic hydroxo moiety similar to that of Glu270 in
carboxypeptidase A. Once the metal-bound hydroxide has formed, it can attack the activated carbonyl carbon,
forming a gem-diolate intermediate that is stabilized by coordination of both oxygen atoms to the co-catalytic
Zn(II) site. The amide nitrogen must also be stabilized, via a hydrogen bond, to make it a suitable leaving group.
This hydrogen bond would also facilitate the collapse of the transition state. The active site glutamate (histidine)
probably supplies the additional proton to the penultimate amino nitrogen, returning it to its ionized state.
Finally, the co-catalytic Zn(II) site releases the cleaved peptides and adds a water molecule that bridges the two
metal ions. Thus, both metal ions are required for full enzymatic activity, but their individual roles appear to
differ markedly.
Several zinc enzymes which catalyse the hydrolysis of phosphoesters have catalytic sites which contain three
metal ions in close proximity. These include (
Figure 12.17
) alkaline phosphatase, phospholipase C and nuclease
P1. In phospholipase C and nuclease P1, all three metal ions are Zn
2
þ
. However the third Zn
2
þ
ion is not directly
associated with the dizinc unit. All three Zn
2
þ
ions are pentacoordinate. Alkaline phosphatase shows structural
similarity to phospholipase C and nuclease P1; however the third metal ion is Mg
2
þ
. One of the Zn
2
þ
sites shares
a common Asp ligand with the Zn
2
þ
site, which is typically hexacoordinate.
Finally, we should briefly mention the purple acid phosphatases, which, unlike the alkaline phosphatases, are
able to hydrolyse phosphate esters at acid pH values. Their purple colour is associated with a Tyr to Fe(III) charge
transfer band. The mammalian purple acid phosphatase is a dinuclear Fe(II)-Fe(III) enzyme, whereas the dinu-
clear site in kidney bean purple acid phosphatase (
Figure 12.13
) has a Zn(II), Fe(III) centre with bridging
