POTASSIUM-DEPENDENT MOLECULES 203
affected by Na + - or K + - dependent conformational changes. A fl uorescent
analog of ATP, TNP - ATP [2 ′ ,3 ′ - O - (2,4,6 - trinitrophenyl)adenosine 5 ′ - triphos-
phate, trisodium salt], was used so that binding could be visualized using fl uo-
rescence spectroscopy. FT - IR spectroscopy was used to show that mutations
did not change the secondary loop structure. The following mutants were
studied: the double mutant I417N - N422A and single mutants R423L, Q482L,
E505Q, F548G, and F548Y. It was found that the dissociation constants for
TNP – ATP binding were signifi cantly increased for nearly all point mutations
but not for ATP binding to the I417N - N422A and E505Q mutants. Previously,
these workers and others had shown that mutations of phe475 and glu446
resulted in inhibition of ATP or TNP – ATP binding to the H 4 – H 5 loop. Molecu-
lar modeling carried out by researchers in reference 12 showed that the aro-
matic ring of phe475 and ATP ’ s adenine ring are parallel to each other at a
distance of 0.3 nm, resulting in π – π stacking interactions between them. Nega-
tively charged glu446 forms a hydrogen bond to the NH 2 donor of the adenos-
ine moiety. Gln482 was shown to stabilize a side chain of glu446 through
hydrogen bonding, thus stabilizing glu446 ’ s position. Phe548 was mutated to
both tyrosine (Y) and glycine (G) with the observations that there was strong
inhibition of ATP binding for the F548Y mutant but weak inhibition for the
F548G mutant. Steric hindrance in the ATP - binding site by the large tyrosine
(Y) residue was invoked to explain this observation. In addition, the authors
observed that phe548, although playing a minor role in direct interaction with
ATP, does help support and maintain the ATP - binding pocket structure. Sur-
prisingly, the R423L mutant showed the strongest inhibition of ATP binding,
although the arginine residue lies outside the ATP - binding pocket. The authors
concluded that the arginine residue forms a strong hydrogen bond with glu472,
and this bonding contributes to pocket stability and orientation of ATP - binding
residues phe475, lys480, and gln482 into their proper positions. The S445A and
E505Q mutations had no signifi cant effect on ATP binding, indicating their
minor role in ATP recognition or binding. In conclusion, the authors showed
that the ATP recognition and binding pocket includes not only the previously
identifi ed important residues of lys480, lys501, gly502, and cys549, but also
includes residues glu446, phe475, gln482, and phe548.
In comparing Na + /K + - ATPase structures to those of other P - type ATPases,
we fi nd that available structures for the Na + /K + - ATPase system are not as
robust and informative as those for Ca 2+ - ATPase. The reader is referred to
Section 6.4.2 for further discussion of the Ca 2+ - ATPase system.
5.4.2 Potassium (K + ) Ion Channels
5.4.2.1 Introduction. Studies on ion channels, potassium ion channels in
particular, carried out by Roderick MacKinnon and his research group, resulted
in his receiving the 2003 Nobel Prize in chemistry “ for structural and mecha-
nistic studies of ion channels. ” Thanks to this contribution and research con-
tinuing in the MacKinnon and many other laboratories, it is now possible to