CHEMXXXX

Catalytic Mechanisms of Binuclear

Metallohydrolases, A Case Study of GpdQ

Name: XXX
Student ID: XXX

Due: 6 June 2014

Catalytic Mechanisms of Binuclear Metallohydrolases,

A Case Study of GpdQ
This paper will focus on Glycerophosphodiesterase (GpdQ), an enzyme from Enterobacter
aerogenes, which is a promising candidate for bioremediation applications. GpdQ is a member of a
group of enzymes knows as binuclear metallohydrolases, which are defined by two metal ions which
catalyse the hydrolysis of amide and ester bonds of carboxylic and phosphoric acids, such as those
found in the pesticides that build up in our environment.
GpdQ can break down pesticides because it has a promiscuous nature in that it can catalyse the
degradation of a variety of phosphodiesters. Phosphodiesters are made up of a phosphate group
between two 5-carbon rings, over two ester bonds. Phosphodiesters are essential to all life on Earth
since they make up the backbone of DNA. As GpdQ is an enzyme that can catalyse the degradation
of a variety of phosphodiesters, it needs to be carefully controlled. Nature designed GpdQ with a
built in safety mechanism: it will only remain active until the substrate is depleted. This prevents side
reactions which may be unwanted (and potentially detrimental to associated in vivo systems) 1.
Chemists seek to understand this mechanism in the hope of creating an enzyme that can be targeted
to a particular substrate (such as a pesticide) without disrupting other side reactions. Their
understanding has come a long way.
Chemists initially established that GpdQ requires two divalent metal ions to function (figure 1).
The site is more tightly bound than the relatively exposed site, so it is predominantly present as
a mononuclear (figure 2) and catalytically inactive enzyme until a preferred substrate is introduced3.
When the substrate enters the vicinity of the site, this sites coordination flexibility allows a
conformational change to take place which increases its affinity for metal ions. Binding with the
substrate causes the bond between the metal and Asn80 to break. The bridging hydroxide then
activates a terminally bound nucleophile via hydrogen bonding interaction 5. This is particularly
interesting because our understanding of the catalytic mechanisms of binuculear metallohydrolases
had previously been based on another enzyme called purple acid phosphatase (PAP). Chemists
understanding of GpdQ indicated a sequential mechanism, rather than PAPs processive mechanism
(see figure 3). Specifically it was thought that GpdQ releases the substrate after cleaving only one
ester bond then cleaves the other ester bond in a second pass, while PAP hydrolyses diester
substrates without releasing an intermediate.
A study2 was conducted to confirm this theory (using 31P NMR) and found that a monoester was
present with use of GpdQ, while measurable quantities were not detected with PAP. Thus, PAP is an
example of an enzyme which utilizes two nucleophiles in sequence and the product of the first
hydrolysis remains associated with the site. On the other hand, GpdQ may regenerate its active site
after the first hydrolysis.
The composition of the metal ions in vivo is also important although unfortunately not currently
known. It is believed that the site prefers Fe(II). It is speculated that site is Fe, Zn or Mn, possibly
Co or Cd 4. Natural abundance rather than catalytic optimisation would suggest Fe and Zn4.
While this paper focused on one particular enzyme, binuclear metallohydrolases are a large and
growing family of enzymes which are involved in many vital reactions. The more we learn about
them, the greater chance we have of harnessing them for practical applications.

Figure 1 : Binuclear metal ion center in GpdQ. The site containing a metal ion is more tightly bound than site. With
the introduction of a substrate, the site undergoes a conformational change which increases its affinity for metal ions,
making it catalytically active.

Figure 2 : Proposed reaction mechanism for GpdQ. Binding with the substrate causes the -Asn80 bond to break, the
bridging hydroxide activates a terminally bound nucleophile.

Figure 3 : Sequential (GpdQ) vs. processive (PAP) mechanism. GpdQ releases the substrate after cleaving only one ester
bond. PAP uses two nucleophiles to cleave the diester bond and the product of the first hydrolysis remains associated
with the site.

References
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