| THE ACTIVE SITE
Thymidine is located in the active site, to undergo phosphorylation.
It sits here in tk Here is one subunit showing a phosphate already transferred to thymidine,
and ADP still bound.
A distinctive feature of HSV-1 is that the nucleoside binding
site is composed of amino acid side chains derived from a set
of almost parallel helices especially from residues 85-89, which
is in helix 2
The quaternary structure has some van der Waals interactions are
shown here in helix 3 (96-108)
In addition, the carboxamide of Gln 125 is in the pocket and forms
hydrogen bonds with the N3, O4-alpha atoms of thymidine. Tyr 172
"stacks" onto the thymidine ring and forms a hydrogen bond to
Arg 163 which binds to PO4 of dTMP. On the opposite side of the
ring, a Met 128 is contacted
| ACTIVE SITE MUTANTS
D162N
Asp 162 (or just simply a negatively charged residue) may be important
for HSV-1 because of one study (Fetzer, 1993). He showed that
an HSV-1 tk mutant D162N lacks enzymatic activity as they were
unable to uncover HSV-1 tk D162N - recombinant vaccinia viruses
(a different simplex of Herpesviradae). What this means is a mutant
was analyzed to find out more about HSV-1 tk. The wild type HSV-1
was compared to a mutant form of vaccinia virus because they are
similiar. What they found was by changing the aspartic acid residue
to an asparagine residue, tk lost enzymatic activity. This is
a relatively new find within the past few years, and I haven't
seen more information about it. The Km of HSV-1 tk and the tk
of vaccinia virus was compared, and this was the result:
Km for HSV-1 tk = 0.05 to 1.0 µm
Km for D162Q tk = .025 to 10 µm
This large difference shows a marked reduction in the ability
of D162Q to bind dT and supports the proposed role of D162Q in
substrate binding the Kcat (turnover) for D162Q.It is reduced
which results in a large difference in the specificity constant
(Kcat/Km) with respect to the HSV-1 tk. This decrease shows minor,
and/or local structural purturbations caused by the substituted
residue (Asn) which may interfere with phosphate transfer and/or
product release.
S1
S1 is another mutant of HSV-1, with only one residue substitution,
where a Tyrosine takes the place of Cysteine at position 336.
Normally, Cys 336 is virutually invisible and located towards
the N-terminus of the protein (residues 335-344) which interacts
with a loop (not the same as the ATP P-loop) preceeding this area.
The substitution of a Tyr for a Cys at position 336 would mark
significat disrutption becaause Tyr is much more bulky than Cys.
It is speculated that local relative displacements would require 1 full atomic
diameter to make room for this ring of Tyr and its distal OH groups.
When tk is muted and Tyr is substituted, it displays extremely
reduced activity towards ATP and the nucleoside. This proves that
Cys 336 is strongly conserved in the particular tk.
| ATP BINDING SITE
The ATP is located in a giant hole which is cradled by residues
56-63. It also accomodates the beta phosphate of ATP. This hole
or specified area is called a P-loop (already shown above). Thr
64 is an important residue because it forms 2 (weak) hydrogen
bonds to ADP-alpha PO4. Also, ATP is at a low specificity, and
tk can accept GTP or CTP as a PO4 source.
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