Background|NNP Ecto-|NNP Nucleoside|NNP triphosphate|NN diphosphohydrolases|NNS (|( E-NTPDases|NNP ,|, formerly|RB called|VBN ecto-|NN ATPases|NNP )|) hydrolyze|NN nucleotides|NNS in|IN the|DT presence|NN of|IN divalent|NN cations|NNS and|CC are|VBP insensitive|JJ to|TO inhibitors|NNS of|IN P-|NNP type|NN ,|, F-|NNP type|NN ,|, and|CC V-|NNP type|NN ATPases|NNP [|NN 1|CD ]|NN .|. 
Three|CD isoforms|NNS that|WDT differ|VBP in|IN the|DT ratio|NN of|IN ATPase|NNP /|NN ADPase|NNP activity|NN are|VBP present|JJ on|IN the|DT cell|NN surface|NN [|NN 2|CD ]|NN :|: E-NTPDase1|NN with|IN a|DT ratio|NN of|IN 1|CD ,|, E-NTPDase2|NN with|IN a|DT ratio|NN of|IN 10|CD and|CC E-NTPDase3|NN with|IN a|DT ratio|NN of|IN 3|CD -|: 5|CD .|. 
NTPDases|NNP are|VBP important|JJ in|IN many|JJ physiological|JJ processes|NNS like|IN cell|NN motility|NN ,|, adhesion|NN ,|, nonsynaptic|JJ information|NN transfer|NN ,|, secretion|NN ,|, regulation|NN of|IN hemostasis|NNS and|CC ectokinases|NNS [|NN 1|CD ]|NN .|. 
Understanding|NNP the|DT enzymatic|JJ mechanisms|NNS of|IN the|DT NTPDases|NNP will|MD help|VB description|NN of|IN their|PRP$ physiological|JJ functions|NNS ,|, and|CC development|NN of|IN strategies|NNS to|TO regulate|VB the|DT functions|NNS of|IN the|DT enzymes|NNS .|. 

The|DT catalytic|JJ mechanism|NN of|IN NTPDases|NNP is|VBZ not|RB known|VBN even|RB though|IN some|DT basic|JJ facts|NNS of|IN the|DT catalysis|NNS have|VBP been|VBN established|VBN .|. 
NTPDases|NNP do|VBP not|RB form|VB phosphorylated|JJ intermediates|NNS during|IN catalysis|NNS ,|, a|DT conclusion|NN also|RB supported|VBN by|IN lack|NN of|IN vanadate|NN sensitivity|NN and|CC Pi|NNP product|NN inhibition|NN [|NN 3|CD 4|CD 5|CD 6|CD ]|NN .|. 
The|DT catalytic|JJ reaction|NN appears|VBZ to|TO be|VB irreversible|JJ and|CC no|DT partial|JJ reactions|NNS have|VBP been|VBN observed|VBN [|NN 7|CD 8|CD ]|NN .|. 
Divalent|NNP cations|NNS like|IN Ca|MD 2|CD +|NN or|CC Mg|NNP 2|CD +|NN are|VBP required|VBN for|IN activity|NN ,|, and|CC maximal|NN activity|NN is|VBZ reached|VBN when|WRB the|DT concentrations|NNS of|IN substrates|NNS and|CC divalent|NN cations|NNS are|VBP equal|JJ [|NN 1|CD ]|NN .|. 
The|DT specific|JJ activities|NNS of|IN NTPDases|NNP vary|VBP over|IN a|DT broad|JJ range|NN from|IN ten|CD thousand|CD units|NNS for|IN potato|NN apyrase|NN to|TO less|JJR than|IN one|CD hundred|CD units|NNS for|IN chicken|NN gizzard|NN ecto-|NN ATPase|NNP [|NN 9|CD 10|CD ]|NN .|. 
Sequence|NNP comparisons|NNS indicate|VBP that|IN most|JJS of|IN NTPDases|NNP contain|VB five|CD highly|RB conserved|JJ regions|NNS ,|, apyrase|NN conserved|JJ region|NN ,|, ACR1|NN -|: ACR5|NN [|NN 9|CD 11|CD ]|NN .|. 
However|RB ,|, the|DT catalytic|JJ sites|NNS have|VBP not|RB been|VBN identified|VBN ,|, although|IN ACR1|NN and|CC ACR4|NN have|VBP been|VBN implicated|VBN in|IN β-|NN and|CC γ-phosphate|JJ binding|JJ ,|, respectively|RB [|NN 9|CD ]|NN .|. 

E-NTPDase1|NN is|VBZ also|RB called|VBN CD39|NN ,|, as|IN it|PRP was|VBD first|RB described|VBD as|IN an|DT antigen|NN present|JJ on|IN activated|VBN B|NNP and|CC T|NN lymphocytes|NNS .|. 
Residues|NNP of|IN ACR1|NN to|TO ACR5|NN of|IN CD39|NN have|VBP been|VBN mutated|VBN to|TO study|VB the|DT involvement|NN of|IN the|DT ACR|NNP regions|NNS in|IN catalysis|NNS .|. 
E174|NN in|IN ACR3|NN and|CC S218|NN in|IN ACR4|NN are|VBP required|VBN for|IN catalytic|JJ function|NN [|NN 12|CD ]|NN .|. 
Substitution|NNP of|IN H59|NN in|IN ACR1|NN converted|VBN CD39|NN into|IN an|DT ADPase|NNP in|IN a|DT quaternary|JJ structure|NN dependent|JJ manner|NN [|NN 13|CD ]|NN .|. 
Mutation|NNP of|IN W187|NN A|DT in|IN ACR3|NN affected|VBN CD39|NN folding|VBG and|CC translocation|NN ,|, while|IN mutation|NN of|IN W459|NN A|DT in|IN ACR5|NN increased|VBN ATPase|NNP activity|NN but|CC diminished|VBN ADPase|NNP activity|NN [|NN 14|CD ]|NN .|. 
Mutations|NNP of|IN D62|NN and|CC G64|NN of|IN ACR1|NN and|CC D219|NN and|CC G221|NN of|IN ACR4|NN demonstrated|VBN that|IN the|DT nucleotide|NN phosphate|NN binding|JJ domains|NNS of|IN NTPDases|NNP are|VBP similar|JJ to|TO those|DT present|NN in|IN the|DT actin|NN /|NN heat|NN shock|NN protein|NN /|NN sugar|NN kinase|NN superfamily|RB [|NN 15|CD ]|NN .|. 
These|DT results|NNS suggest|VBP that|IN the|DT conserved|JJ residues|NNS of|IN the|DT ACR1|NN to|TO 5|CD regions|NNS are|VBP involved|VBN in|IN the|DT catalytic|JJ mechanism|NN of|IN CD39|NN .|. 

The|DT catalytic|JJ activity|NN of|IN CD39|NN is|VBZ dependent|JJ on|IN the|DT presence|NN of|IN divalent|NN cations|NNS .|. 
Since|IN the|DT interactions|NNS of|IN Ca|MD +|NN 2|CD and|CC Mg|NNP +|NN 2|CD with|IN proteins|NNS are|VBP difficult|JJ to|TO study|VB due|JJ to|TO the|DT lack|NN of|IN spectroscopic|JJ properties|NNS ,|, vanadyl|NN (|( V|NNP IV|NNP =|SYM O|NNP )|) 2|CD +|NN has|VBZ been|VBN used|VBN as|IN a|DT probe|NN of|IN the|DT ligands|NNS that|WDT compose|NN Mg|NNP 2|CD +|NN ,|, Ca|MD 2|CD +|NN ,|, and|CC Mn|NNP 2|CD +|NN binding|JJ sites|NNS of|IN several|JJ proteins|NNS ,|, including|VBG carboxypeptidase|NN [|NN 16|CD ]|NN ,|, S-|NNP adenosylmethionine|NN synthetase|NN [|NN 17|CD 18|CD ]|NN ,|, pyruvate|NN kinase|NN [|NN 19|CD 20|CD ]|NN ,|, and|CC F|NN 1|CD -|: ATPase|NNP [|NN 21|CD 22|CD ]|NN .|. 
This|DT cation|NN specifically|RB binds|NNS to|TO divalent|NN cation|NN binding|JJ sites|NNS of|IN several|JJ enzymes|NNS ,|, and|CC in|IN many|JJ cases|NNS serves|VBZ as|IN a|DT functional|JJ cofactor|NN [|NN 23|CD ]|NN .|. 
Vanadyl|NNP has|VBZ one|CD axial|NN and|CC four|CD equatorial|NN coordination|NN sites|NNS relative|JJ to|TO the|DT axis|NNS of|IN the|DT double-bounded|JJ oxygen|NN ,|, an|DT arrangement|NN that|WDT is|VBZ similar|JJ to|TO that|DT for|IN Ca|MD 2|CD +|NN and|CC Mg|NNP 2|CD +|NN .|. 
As|IN it|PRP is|VBZ known|VBN that|IN the|DT A|DT and|CC g|SYM tensors|NNS derived|VBN from|IN the|DT EPR|NNP spectrum|NN of|IN bound|VBN VO|NNP 2|CD +|NN are|VBP a|DT direct|JJ measure|NN of|IN the|DT nature|NN of|IN the|DT equatorial|NN metal|NN ligands|NNS [|NN 24|CD ]|NN ,|, binding|JJ of|IN VO|NNP 2|CD +|NN to|TO CD39|NN could|MD provide|VB details|NNS about|IN the|DT catalytic|JJ mechanism|NN of|IN CD39|NN .|. 

Recently|RB we|PRP reported|VBD that|IN a|DT recombinant|JJ soluble|JJ CD39|NN ,|, capable|JJ of|IN hydrolyzing|VBG both|DT ATP|NNP and|CC ADP|NNP ,|, was|VBD expressed|VBN and|CC purified|JJ from|IN insect|NN cells|NNS [|NN 25|CD ]|NN .|. 
Only|RB one|CD nucleotide-binding|JJ site|NN was|VBD identified|VBN on|IN the|DT purified|JJ soluble|JJ CD39|NN in|IN the|DT presence|NN of|IN Ca|MD 2|CD +|NN when|WRB non-hydrolysable|JJ nucleotide|NN analogs|NNS were|VBD used|VBN .|. 
In|IN this|DT report|NN ,|, we|PRP characterized|VBD the|DT signals|NNS that|WDT were|VBD obtained|VBN from|IN bound|VBN VO|NNP 2|CD +|NN when|WRB ATP|NNP or|CC ADP|NNP was|VBD present|JJ at|IN the|DT catalytic|JJ site|NN of|IN the|DT purified|JJ soluble|JJ CD39|NN .|. 
The|DT possible|JJ metal|NN ligands|NNS for|IN VO|NNP +|NN 2|CD at|IN the|DT catalytic|JJ site|NN are|VBP proposed|VBN and|CC the|DT catalytic|JJ mechanism|NN is|VBZ discussed|VBN .|. 

Results|NNS Nucleotidase|NNP activity|NN of|IN purified|JJ soluble|JJ CD39|NN with|IN VO|NNP 2|CD +|NN as|IN cofactor|NN The|DT ability|NN of|IN purified|JJ soluble|JJ CD39|NN to|TO hydrolyze|NN VO|NNP 2|CD +|NN ATP|NNP is|VBZ shown|VBN in|IN Figure|NN 1|CD .|. Soluble|NNP CD39|NN did|VBD not|RB hydrolyze|NN either|CC ATP|NNP or|CC ADP|NNP in|IN the|DT absence|NN of|IN VO|NNP 2|CD +|NN (|( Fig.|NN 1|CD A|DT )|) .|. 
When|WRB VO|NNP 2|CD +|NN was|VBD mixed|VBN with|IN ATP|NNP at|IN a|DT ratio|NN of|IN 1|CD :|: 1|CD ,|, the|DT concentrations|NNS of|IN both|DT ADP|NNP and|CC AMP|NNP increased|VBD and|CC ATP|NNP decreased|VBD as|IN the|DT incubation|NN time|NN was|VBD prolonged|JJ (|( Fig.|NN 1|CD B|NNP )|) .|. 
The|DT ATPase|NNP activity|NN of|IN sCD39|NN with|IN VO|NNP 2|CD +|NN was|VBD about|IN 25|CD %|NN of|IN that|DT with|IN Ca|MD 2|CD +|NN as|IN a|DT cofactor|NN .|. 
Vanadyl|NNP is|VBZ unstable|JJ in|IN aqueous|JJ solution|NN at|IN pH7.0|NN in|IN the|DT absence|NN of|IN chelator|NN and|CC will|MD precipitate|NN out|IN of|IN solution|NN as|IN [|NN VO|NNP (|( OH|NNP )|) 2|CD ]|NN n|NN .|. 
The|DT rate|NN of|IN precipitation|NN depends|VBZ on|IN the|DT abundance|NN and|CC affinity|NN of|IN the|DT chelator|NN .|. 
This|DT means|VBZ that|IN the|DT actual|JJ VO|NNP 2|CD +|NN concentration|NN was|VBD lower|JJR than|IN 0.5|CD mM|NN .|. 
This|DT result|NN indicates|VBZ that|IN VO|NNP 2|CD +|NN can|MD functionally|RB substitute|VB for|IN Ca|MD 2|CD +|NN as|IN cofactor|NN for|IN sCD39|NN nucleotidase|NN activity|NN .|. 

Characterization|NNP of|IN bound|VBN VO|NNP 2|CD +|NN ADPNP|NNP by|IN CW-EPR|NNP The|DT parallel|JJ features|NNS of|IN CW-EPR|NNP spectrum|NN of|IN bound|VBN VO|NNP 2|CD +|NN in|IN the|DT presence|NN of|IN ADPNP|NNP ,|, an|DT ATP|NNP analog|NN ,|, are|VBP shown|VBN in|IN Figure|NN 2|CD a|DT .|. 
This|DT spectrum|NN shows|VBZ 51|CD V|NNP hyperfine|NN splitting|NN and|CC the|DT center|NN of|IN the|DT parallel|JJ transitions|NNS from|IN molecules|NNS with|IN the|DT V|NNP =|SYM O|NNP bond|NN oriented|VBN along|IN the|DT magnetic|JJ field|NN (|( A|DT ||NN ||NN ,|, g|SYM ||NN ||NN )|) which|WDT are|VBP strong|JJ enough|RB to|TO tell|VB the|DT nature|NN of|IN VO|NNP 2|CD +|NN equatorial|NN ligands|NNS [|NN 22|CD ]|NN .|. 
Of|IN the|DT eight|CD transitions|NNS that|WDT result|NN from|IN the|DT parallel|RB oriented|VBN molecules|NNS ,|, the|DT -|: 7/2|CD ||NN ||NN ,|, -|: 5/2|CD ||NN ||NN ,|, +|NN 3/2|CD ||NN ||NN ,|, +|NN 5/2|CD ||NN ||NN ,|, and|CC +|NN 7/2|CD ||NN ||NN transitions|NNS (|( shown|VBN in|IN the|DT figures|NNS from|IN left|VBD to|TO right|NN ,|, respectively|RB )|) do|VBP not|RB overlap|VB with|IN perpendicular|NN transitions|NNS .|. 
The|DT 51|CD V|NNP hyperfine|NN splitting|NN spectra|NN from|IN molecules|NNS with|IN V|NNP =|SYM O|NNP bond|NN perpendicular|NN to|TO the|DT magnetic|JJ field|NN (|( A|DT ⊥|NN )|) are|VBP much|RB smaller|JJR and|CC not|RB shown|VBN here|RB [|NN 21|CD ]|NN .|. 
The|DT intensity|NN of|IN -|: 5/2|CD ||NN ||NN peak|NN is|VBZ used|VBN as|IN direct|JJ measurement|NN of|IN the|DT amount|NN of|IN bound|VBN VO|NNP 2|CD +|NN ,|, since|IN this|DT peak|NN is|VBZ the|DT most|RBS intense|JJ peak|NN in|IN the|DT EPR|NNP spectrum|NN that|WDT contains|VBZ contribution|NN only|RB from|IN A|DT ||NN ||NN but|CC not|RB A|DT ⊥|NN [|NN 21|CD 26|CD ]|NN .|. 
In|IN this|DT study|NN ,|, the|DT intensities|NNS of|IN each|DT bound|VBN VO|NNP 2|CD +|NN -|: EPR|NNP feature|NN were|VBD normalized|JJ to|TO 1|CD mg|NN of|IN protein|NN .|. 
VO|NNP 2|CD +|NN bound|VBN as|IN the|DT VO|NNP 2|CD +|NN -|: AMPPNP|NNP complex|JJ to|TO sCD39|NN produced|VBD a|DT strong|JJ spectrum|NN characterized|VBN by|IN A|DT ||NN ||NN of|IN 504.25|CD MHz|NNP and|CC g|SYM ||NN ||NN of|IN 1.9410|CD (|( Fig.|NN 2|CD b|SYM )|) ,|, called|VBN species|NNS T|NN (|( Table|NNP 1|CD )|) .|. 
The|DT best|JJS fit|NN of|IN EPR|NNP species|NNS T|NN to|TO eq|NN 1|CD is|VBZ one|CD equatorial|NN nitrogen|NN from|IN an|DT amino|JJ group|NN and|CC three|CD equatorial|NN oxygen|NN ligands|NNS from|IN carboxyl|NN or|CC phosphate|NN groups|NNS (|( Table|NNP 2|CD )|) .|. 
This|DT result|NN is|VBZ consistent|JJ with|IN AMPPNP|NNP binding|JJ strongly|RB to|TO a|DT single|JJ site|NN on|IN sCD39|NN in|IN the|DT presence|NN of|IN metal|NN [|NN 25|CD ]|NN .|. 

Characterization|NNP of|IN EPR|NNP species|NNS from|IN VO|NNP 2|CD +|NN -|: AMPCP|NNP bound|VBN to|TO sCD39|NN Figure|NN 3|CD ashows|NNS the|DT parallel|JJ features|NNS of|IN the|DT EPR|NNP spectrum|NN of|IN sCD39|NN bound|VBN VO|NNP 2|CD +|NN -|: AMPCP|NNP .|. 
Two|CD sets|NNS of|IN parallel|JJ transitions|NNS were|VBD observed|VBN ,|, and|CC the|DT derived|VBD A|DT ||NN ||NN and|CC g|SYM ||NN ||NN values|NNS are|VBP listed|VBN in|IN Table|NNP 1|CD 
.|. One|CD set|VBN had|VBD A|DT ||NN ||NN of|IN 521.78|CD MHz|NNP and|CC g|SYM ||NN ||NN of|IN 1.937|CD ,|, which|WDT is|VBZ defined|VBN as|IN species|NNS D1|NN (|( Fig.|NN 3|CD b|SYM )|) .|. 
The|DT other|JJ set|VBN displayed|VBD A|DT ||NN ||NN of|IN 490.01|CD MHz|NNP and|CC g|SYM ||NN ||NN of|IN 1.9435|CD ,|, which|WDT is|VBZ called|VBN species|NNS D2|NN (|( Fig.|NN 3|CD c|SYM )|) .|. 
The|DT intensity|NN of|IN species|NNS D1|NN accounted|VBD for|IN 11.4|CD %|NN of|IN species|NNS T|NN from|IN bound|VBN VO|NNP 2|CD +|NN -|: AMPPNP|NNP ,|, and|CC the|DT intensity|NN of|IN D2|NN accounted|VBD for|IN 7.1|CD %|NN of|IN species|NNS T.|NN 
The|DT intensity|NN ratio|NN of|IN species|NNS D1|NN over|IN D2|NN was|VBD 1.6|CD .|. 
In|IN order|NN to|TO distinguish|VB species|NNS D1|NN from|IN D2|NN ,|, the|DT sample|NN with|IN VO|NNP 2|CD +|NN -|: AMPCP|NNP bound|VBN to|TO sCD39|NN was|VBD thawed|JJ and|CC incubated|JJ at|IN room|NN temperature|NN for|IN 30|CD minutes|NNS ,|, and|CC the|DT VO|NNP 2|CD +|NN EPR|NNP spectrum|NN was|VBD collected|VBN again|RB .|. 
As|IN shown|VBN in|IN Figure|NN 4|CD aand|NN 4|CD b|SYM ,|, either|CC A|DT ||NN ||NN or|CC g|SYM ||NN ||NN values|NNS for|IN both|DT species|NNS D1|NN and|CC D2|NN were|VBD changed|VBN .|. 
The|DT intensity|NN of|IN species|NNS D1|NN was|VBD not|RB changed|JJ as|IN it|PRP accounted|VBD for|IN 12.1|CD %|NN of|IN the|DT intensity|NN of|IN species|NNS T|NN of|IN the|DT bound|VBN VO|NNP 2|CD +|NN -|: AMPPNP|NNP .|. 
However|RB ,|, the|DT intensity|NN of|IN species|NNS D2|NN was|VBD decreased|VBN dramatically|RB ,|, and|CC it|PRP accounted|VBD for|IN only|RB 0.1|CD %|NN of|IN the|DT intensity|NN of|IN species|NNS T.|NN 
The|DT intensity|NN ratio|NN of|IN D1|NN over|IN D2|NN increased|VBN about|IN 75|CD fold|VB to|TO become|VB 120|CD .|. 

There|EX are|VBP two|CD sets|NNS of|IN equatorial|NN ligands|NNS that|WDT can|MD fit|VB well|RB the|DT EPR|NNP species|NNS D1|NN according|VBG to|TO Eq|NNP 1|CD (|( Table|NNP 2|CD )|) .|. 
One|CD set|VBN includes|VBZ two|CD equatorial|NN oxygen|NN from|IN two|CD water|NN molecules|NNS ,|, one|CD equatorial|NN oxygen|NN from|IN a|DT carboxyl|NN group|NN or|CC phosphate|NN ,|, and|CC one|CD equatorial|NN nitrogen|NN from|IN an|DT amino|JJ group|NN .|. 
The|DT other|JJ set|VBN contains|VBZ one|CD equatorial|NN oxygen|NN from|IN water|NN and|CC three|CD equatorial|NN oxygens|NNS from|IN carboxyl|NN groups|NNS or|CC phosphate|NN .|. 
The|DT best|JJS fit|NN for|IN the|DT EPR|NNP species|NNS D2|NN to|TO eq|NN 1|CD is|VBZ one|CD equatorial|NN oxygen|NN from|IN a|DT hydroxyl|NN group|NN and|CC three|CD equatorial|NN oxygens|NNS from|IN carboxyl|NN groups|NNS or|CC phosphate|NN .|. 

EPR|NNP characteristics|NNS of|IN sCD39|NN bound|VBN VO|NNP 2|CD +|NN -|: ATP|NNP In|IN order|NN to|TO capture|VB the|DT bound|VBN VO|NNP 2|CD +|NN -|: EPR|NNP signal|NN before|IN the|DT enzyme|NN completely|RB turned|VBD over|IN ,|, sCD39|NN and|CC VO|NNP 2|CD +|NN -|: ATP|NNP were|VBD mixed|JJ on|IN ice|NN ,|, immediately|RB transferred|VBN into|IN the|DT EPR|NNP tube|NN and|CC frozen|VBN .|. 
The|DT entire|JJ process|NN took|VBD about|IN 15|CD seconds|NNS .|. 
The|DT parallel|JJ portion|NN of|IN the|DT collected|VBN VO|NNP 2|CD +|NN -|: EPR|NNP spectrum|NN is|VBZ shown|VBN in|IN Figure|NN 5|CD a|DT .|. 
VO|NNP 2|CD +|NN -|: ATP|NNP complex|JJ bound|VBN to|TO sCD39|NN produced|VBD an|DT EPR|NNP spectrum|NN with|IN A|DT ||NN ||NN of|IN 489.5|CD MHz|NNP and|CC g|SYM ||NN ||NN of|IN 1.9455|CD ,|, which|WDT corresponded|VBD to|TO species|NNS D2|NN (|( Fig.|NN 5|CD b|SYM )|) .|. 
The|DT signal|NN intensity|NN from|IN the|DT bound|VBN VO|NNP 2|CD +|NN -|: nucleotide|NN complex|NN accounted|VBD only|RB for|IN 7.5|CD %|NN of|IN that|DT of|IN species|NNS T|NN from|IN bound|VBN non-hydrolysable|JJ VO|NNP 2|CD +|NN -|: ADPNP|NNP complex|NN .|. 

The|DT same|JJ sample|NN made|VBN from|IN mixing|VBG VO|NNP 2|CD +|NN -|: ATP|NNP and|CC sCD39|NN was|VBD incubated|JJ at|IN room|NN temperature|NN for|IN 30|CD minutes|NNS ,|, then|RB the|DT VO|NNP 2|CD +|NN -|: EPR|NNP spectrum|NN was|VBD generated|VBN as|IN shown|VBN in|IN Figures|NNS 4|CD cand|NN 4|CD d|SYM .|. 
The|DT EPR|NNP parameters|NNS derived|VBN from|IN this|DT VO|NNP 2|CD +|NN -|: EPR|NNP spectrum|NN were|VBD 489.5|CD MHz|NNP for|IN A|DT ||NN ||NN and|CC 1.9455|CD for|IN g|SYM ||NN ||NN respectively|RB ,|, which|WDT is|VBZ consistent|JJ with|IN species|NNS D2|NN .|. 
However|RB ,|, the|DT signal|NN intensity|NN decreased|VBD about|IN 37.5|CD 
fold|VB compared|VBN to|TO that|DT obtained|VBD before|IN room|NN temperature|NN incubation|NN .|. 

Free|NNP VO|NNP 2|CD +|NN binding|JJ to|TO sCD39|NN characterized|VBN by|IN CD-EPR|NNP Like|IN other|JJ metals|NNS (|( Ca|MD 2|CD +|NN and|CC Mg|NNP 2|CD +|NN )|) ,|, free|JJ VO|NNP 2|CD +|NN inhibited|VBD the|DT nucleotidase|NN activities|NNS of|IN sCD39|NN at|IN high|JJ concentration|NN (|( data|NNS not|RB shown|VBN )|) .|. 
VO|NNP 2|CD +|NN in|IN the|DT absence|NN of|IN any|DT nucleotides|NNS was|VBD added|VBN to|TO sCD39|NN at|IN 1|CD :|: 1|CD molar|NN ratio|NN .|. 
The|DT parallel|JJ transitions|NNS of|IN bound|VBN VO|NNP 2|CD +|NN -|: EPR|NNP spectrum|NN are|VBP shown|VBN in|IN Figure|NN 6|CD .|. 
The|DT features|NNS derived|VBN from|IN the|DT VO|NNP 2|CD +|NN -|: EPR|NNP spectrum|NN were|VBD 486|CD MHz|NNP for|IN A|DT ||NN ||NN and|CC 1.946|CD for|IN g|SYM ||NN ||NN ,|, which|WDT was|VBD designed|VBN as|IN species|NNS V|NNP (|( Fig.|NN 6|CD b|SYM )|) .|. 
The|DT signal|NN intensity|NN of|IN bound|VBN VO|NNP 2|CD +|NN accounted|VBD for|IN 20.3|CD %|NN of|IN that|DT from|IN the|DT bound|VBN VO|NNP 2|CD +|NN -|: ADPNP|NNP complex|NN .|. 
The|DT best|JJS fit|NN of|IN equatorial|NN ligands|NNS for|IN species|NNS V|NNP according|VBG to|TO eq|NN 1|CD is|VBZ two|CD equatorial|NN oxygen|NN from|IN hydroxyl|NN groups|NNS and|CC another|DT two|CD equatorial|NN oxygen|NN from|IN two|CD water|NN molecules|NNS .|. 

Discussion|NNP Vanadyl|NNP has|VBZ been|VBN used|VBN to|TO estimate|VB the|DT types|NNS of|IN groups|NNS that|WDT serve|VBP as|IN metal-ligands|JJ in|IN F|NN 1|CD -|: ATPase|NNP and|CC other|JJ enzymes|NNS [|NN 16|CD 18|CD 19|CD 21|CD ]|NN because|IN the|DT g|SYM and|CC A|DT tensors|NNS of|IN the|DT 51|CD V|NNP hyperfine|NN couplings|NNS are|VBP approximately|RB a|DT linear|JJ combination|NN of|IN tensors|NNS from|IN each|DT type|NN of|IN group|NN that|WDT contributes|VBZ an|DT equatorial|NN ligand|NN [|NN 24|CD 27|CD ]|NN .|. 
By|IN studying|VBG the|DT EPR|NNP spectra|NN of|IN bound|VBN VO|NNP 2|CD +|NN in|IN the|DT presence|NN of|IN different|JJ nucleotides|NNS ,|, we|PRP show|VBP that|IN the|DT interaction|NN of|IN soluble|JJ CD39|NN with|IN ATP|NNP is|VBZ different|JJ from|IN that|DT with|IN ADP|NNP .|. 

It|PRP is|VBZ not|RB surprising|JJ that|IN VO|NNP 2|CD +|NN can|MD functionally|RB replace|VB Ca|MD 2|CD +|NN in|IN the|DT hydrolysis|NNS of|IN both|DT ATP|NNP and|CC ADP|NNP by|IN soluble|JJ CD39|NN ,|, although|IN the|DT enzymatic|JJ activity|NN is|VBZ about|IN 25|CD %|NN of|IN that|DT with|IN Ca|MD 2|CD +|NN as|IN the|DT cofactor|NN ,|, since|IN F|NN 1|CD -|: ATPase|NNP also|RB hydrolyzes|NNS ATP|NNP at|IN a|DT decreased|VBN rate|NN when|WRB VO|NNP 2|CD +|NN replaces|VBZ Mg|NNP 2|CD +|NN [|NN 21|CD ]|NN .|. 

The|DT EPR|NNP features|NNS of|IN VO|NNP 2|CD +|NN are|VBP able|JJ to|TO reveal|VB some|DT details|NNS about|IN how|WRB CD39|NN hydrolyzes|NNS ATP|NNP and|CC ADP|NNP .|. 
A|DT single|JJ EPR|NNP feature|NN ,|, species|NNS T|NN ,|, was|VBD observed|VBN when|WRB ADPNP|NNP (|( a|DT non-hydrolyzable|JJ analog|NN of|IN ATP|NNP )|) complexed|JJ with|IN VO|NNP 2|CD +|NN was|VBD bound|VBN to|TO sCD39|NN ,|, which|WDT is|VBZ consistent|JJ with|IN the|DT presence|NN of|IN only|RB one|CD nucleotide|NN binding|JJ site|NN [|NN 25|CD ]|NN .|. 
The|DT g|SYM and|CC A|DT tensors|NNS derived|VBN from|IN species|NNS T|NN are|VBP 1.9410|CD and|CC 504.25|CD MHz|NNP respectively|RB ,|, which|WDT can|MD be|VB fitted|JJ best|JJS with|IN one|CD amino|JJ group|NN and|CC three|CD groups|NNS combined|VBN from|IN carboxyl|NN and|CC phosphate|NN groups|NNS as|IN the|DT equatorial|NN ligands|NNS of|IN the|DT bound|VBN VO|NNP 2|CD +|NN on|IN sCD39|NN .|. 
In|IN accordance|NN with|IN metal-|NN ATP|NNP complex|JJ coordination|NN on|IN other|JJ enzymes|NNS that|WDT hydrolyze|NN ATP|NNP ,|, like|IN F|NN 1|CD -|: ATPase|NNP [|NN 22|CD 28|CD ]|NN ,|, the|DT γ-|NN and|CC β-phosphate|JJ of|IN ATP|NNP most|RBS likely|JJ bind|NN to|TO VO|NNP 2|CD +|NN while|IN the|DT third|JJ carboxyl|NN group|NN is|VBZ contributed|VBN by|IN a|DT side-chain|JJ of|IN aspartate|NN or|CC glutamate|NN of|IN sCD39|NN .|. 
It|PRP is|VBZ not|RB unusual|JJ for|IN the|DT ε-amino|JJ group|NN of|IN lysine|NN to|TO coordinate|VB with|IN metals|NNS in|IN enzymes|NNS .|. 
It|PRP has|VBZ been|VBN reported|VBN that|IN the|DT amino|JJ group|NN serves|VBZ as|IN one|CD of|IN VO|NNP 2|CD +|NN equatorial|NN ligands|NNS in|IN CF|NNP 1|CD -|: ATPase|NNP [|NN 21|CD ]|NN ,|, pyruvate|NN kinase|NN [|NN 19|CD 20|CD ]|NN ,|, AdoMet|NNP synthetase|NN [|NN 17|CD 18|CD ]|NN ,|, and|CC carboxypeptidase|NN [|NN 16|CD ]|NN .|. 
Thus|RB one|CD amino|JJ group|NN from|IN lysine|NN ,|, one|CD carboxyl|NN group|NN from|IN aspartate|NN or|CC glutamate|NN ,|, and|CC two|CD oxygens|NNS from|IN the|DT phosphates|NNS of|IN ADPNP|NNP serve|VB as|IN the|DT equatorial|NN ligands|NNS of|IN sCD39|NN bound|VBN VO|NNP 2|CD +|NN in|IN the|DT presence|NN of|IN ADPNP|NNP .|. 

In|IN the|DT presence|NN of|IN AMPCP|NNP ,|, bound|VBN VO|NNP 2|CD +|NN produced|VBD two|CD EPR|NNP features|NNS ,|, species|NNS D1|NN and|CC species|NNS D2|NN that|WDT are|VBP separated|VBN by|IN about|IN 30|CD MHz|NNP .|. 
As|IN we|PRP have|VBP reported|VBN that|DT sCD39|NN releases|NNS intermediate|JJ ADP|NNP before|IN ADP|NNP is|VBZ further|JJ cleaved|JJ during|IN ATP|NNP hydrolysis|NNS [|NN 25|CD ]|NN ,|, sCD39|NN probably|RB has|VBZ two|CD conformations|NNS that|WDT bind|NN metal-|NN ADP|NNP complexes|NNS ,|, one|CD is|VBZ the|DT conformation|NN that|IN releases|VBZ the|DT ADP|NNP intermediate|JJ ,|, and|CC another|DT that|WDT recruits|VBZ intermediate|JJ ADP|NNP back|RB to|TO the|DT enzyme|NN for|IN further|JJ hydrolysis|NNS to|TO AMP|NNP .|. 
However|RB ,|, intact|JJ CD39|NN does|VBZ not|RB release|VB intermediate|JJ ADP|NNP during|IN ATP|NNP hydrolysis|NNS ,|, suggesting|VBG that|IN there|EX is|VBZ only|RB one|CD ADP|NNP binding|JJ site|NN on|IN each|DT CD39|NN monomer|NN in|IN the|DT intact|JJ protein|NN [|NN 2|CD 25|CD ]|NN .|. 
The|DT two|CD EPR|NNP species|NNS observed|VBD with|IN VO|NNP 2|CD +|NN -|: AMPCP|NNP probably|RB correspond|VB to|TO the|DT two|CD different|JJ conformations|NNS of|IN bound|VBN ADP|NNP at|IN the|DT same|JJ catalytic|JJ site|NN on|IN sCD39|NN .|. 
The|DT signal|NN intensity|NN of|IN the|DT bound|VBN VO|NNP 2|CD +|NN -|: AMPCP|NNP EPR|NNP spectrum|NN indicates|VBZ that|IN species|NNS D1|NN is|VBZ dominant|JJ over|IN species|NNS D2|NN .|. 
In|IN order|NN to|TO further|VB assign|VB species|NNS D1|NN and|CC D2|NN to|TO the|DT two|CD different|JJ conformations|NNS ,|, two|CD experiments|NNS were|VBD done|VBN (|( Fig.|NN 5|CD )|) .|. 
Incubation|NNP of|IN sCD39|NN with|IN VO|NNP 2|CD +|NN -|: AMPCP|NNP at|IN room|NN temperature|NN resulted|VBD in|IN a|DT dramatic|JJ decrease|NN of|IN the|DT intensity|NN of|IN species|NNS D2|NN ,|, while|IN the|DT signal|NN intensity|NN of|IN species|NNS D1|NN remained|VBD unchanged|JJ .|. 
These|DT data|NNS indicate|VBP that|IN VO|NNP 2|CD +|NN -|: AMPCP|NNP was|VBD released|VBN from|IN the|DT conformation|NN corresponding|JJ to|TO species|NNS D2|NN ;|: however|RB ,|, the|DT conformation|NN corresponding|JJ to|TO species|NNS D1|NN still|RB had|VBD bound|VBN VO|NNP 2|CD +|NN -|: AMPCP|NNP .|. 
More|JJR evidence|NN for|IN two|CD conformations|NNS of|IN the|DT enzyme|NN was|VBD obtained|VBN from|IN the|DT EPR|NNP spectra|NN of|IN bound|VBN VO|NNP 2|CD +|NN -|: ATP|NNP .|. 
No|DT species|NNS T|NN was|VBD found|VBN presumably|RB because|IN ATP|NNP was|VBD converted|VBN to|TO ADP|NNP before|IN the|DT sample|NN was|VBD frozen|VBN .|. 
Only|RB species|NNS D2|NN was|VBD observed|VBN and|CC its|PRP$ intensity|NN decreased|VBD as|IN the|DT incubation|NN time|NN was|VBD prolonged|JJ .|. 
We|PRP suggest|VBP that|IN species|NNS D2|NN corresponds|NNS to|TO the|DT conformation|NN that|IN releases|NNS ADP|NNP as|IN an|DT intermediate|JJ product|NN and|CC species|NNS D1|NN corresponds|NNS to|TO the|DT conformation|NN that|IN binds|NNS ADP|NNP as|IN a|DT substrate|NN .|. 
The|DT lower|JJR signal|NN intensities|NNS of|IN species|NNS D1|NN and|CC D2|NN compared|VBN to|TO that|DT of|IN species|NNS T|NN suggest|VBP that|IN the|DT affinity|NN of|IN sCD39|NN for|IN ADP|NNP or|CC its|PRP$ analog|NN AMPCP|NNP is|VBZ lower|JJR than|IN that|DT for|IN the|DT ATP|NNP analog|NN ADPNP|NNP ,|, which|WDT is|VBZ consistent|JJ with|IN the|DT result|NN that|IN only|RB ATP|NNP analogs|NNS were|VBD detected|VBN on|IN sCD39|NN [|NN 25|CD ]|NN .|. 

The|DT calculated|VBN g|SYM ||NN ||NN and|CC A|DT ||NN ||NN values|NNS that|IN best|RBS matched|VBD the|DT experimental|JJ values|NNS for|IN species|NNS D2|NN suggest|VBP that|IN one|CD hydroxyl|NN group|NN and|CC three|CD oxygens|NNS derived|VBN from|IN carboxyl|NN groups|NNS and|CC phosphates|NNS are|VBP the|DT equatorial|NN ligands|NNS of|IN bound|VBN VO|NNP 2|CD +|NN -|: ADP|NNP .|. 
Since|IN the|DT conformation|NN corresponding|JJ to|TO species|NNS D2|NN is|VBZ found|VBN in|IN the|DT presence|NN of|IN ATP|NNP and|CC is|VBZ likely|JJ to|TO be|VB the|DT conformation|NN that|IN releases|NNS bound|VBN VO|NNP +|NN 2|CD -|: ADP|NNP ,|, it|PRP is|VBZ likely|JJ that|IN the|DT VO|NNP +|NN 2|CD ligands|NNS are|VBP one|CD phosphate|NN and|CC two|CD carboxyl|NN groups|NNS [|NN 25|CD ]|NN .|. 
When|WRB ADP|NNP is|VBZ the|DT substrate|NN and|CC generates|VBZ species|NNS D1|NN ,|, one|CD water|NN molecule|NN and|CC a|DT combination|NN of|IN three|CD groups|NNS between|IN carboxyl|NN groups|NNS and|CC phosphates|NNS serve|VBP as|IN the|DT equatorial|NN ligands|NNS of|IN bound|VBN VO|NNP 2|CD +|NN on|IN sCD39|NN .|. 
The|DT probable|JJ combination|NN of|IN carboxyl|NN groups|NNS and|CC phosphates|NNS for|IN species|NNS D1|NN is|VBZ one|CD carboxyl|NN group|NN and|CC two|CD phosphates|NNS since|IN VO|NNP 2|CD +|NN complexes|NNS ADP|NNP through|IN two|CD phosphates|NNS before|IN VO|NNP 2|CD +|NN -|: ADP|NNP is|VBZ bound|VBN to|TO the|DT enzyme|NN .|. 

The|DT site|NN directed|VBD mutagenesis|NNS studies|NNS on|IN CD39|NN and|CC other|JJ members|NNS of|IN the|DT CD39|NN family|NN give|VBP some|DT hints|NNS about|IN the|DT possible|JJ residues|NNS that|WDT serve|VBP as|IN metal|NN ligands|NNS at|IN the|DT catalytic|JJ site|NN of|IN sCD39|NN .|. 
The|DT changes|NNS of|IN D62|NN on|IN ACR1|NN ,|, E174|NN on|IN ACR3|NN ,|, D213|NN (|( D219|NN in|IN HB6|NN )|) and|CC S218|NN on|IN ACR4|NN dramatically|RB decrease|VB both|DT ATPase|NNP and|CC ADPase|NNP activities|NNS of|IN CD39|NN [|NN 12|CD 15|CD ]|NN .|. 
Figure|NN 7|CD summarizes|NNS the|DT possible|JJ coordination|NN of|IN Ca|MD 2|CD +|NN from|IN the|DT data|NNS of|IN species|NNS T|NN ,|, species|NNS D1|NN ,|, and|CC species|NNS D2|NN in|IN the|DT different|JJ situations|NNS of|IN sCD39|NN catalysis|NNS .|. 
The|DT catalytic|JJ base|NN attack|NN results|NNS in|IN cleavage|NN of|IN the|DT γ-phosphate|JJ of|IN ATP|NNP ,|, and|CC one|CD carboxyl|NN group|NN replaces|VBZ the|DT γ-phosphate|JJ as|IN a|DT metal|NN ligand|NN (|( from|IN species|NNS T|NN to|TO species|NNS D2|NN )|) ,|, which|WDT is|VBZ accompanied|VBN by|IN a|DT swap|NN of|IN an|DT amino|JJ group|NN with|IN a|DT hydroxyl|NN group|NN (|( S218|NN ?|. )|) 
.|. This|DT hydroxyl|NN group|NN (|( S218|NN ?|. )|) probably|RB interacts|NNS with|IN the|DT water|NN molecule|NN through|IN hydrogen|NN bond|NN in|IN the|DT conformation|NN corresponding|JJ to|TO species|NNS D1|NN to|TO hydrolyze|NN ADP|NNP .|. 
The|DT constant|JJ carboxyl|NN group|NN that|WDT appears|VBZ in|IN all|DT conformations|NNS of|IN sCD39|NN hydrolysis|NNS is|VBZ likely|RB contributed|VBN by|IN D213|NN since|IN it|PRP is|VBZ close|JJ to|TO S218|NN .|. 

The|DT results|NNS presented|VBN here|RB also|RB provide|VB an|DT explanation|NN to|TO the|DT free|JJ metal|NN inhibition|NN of|IN CD39|NN catalytic|JJ activity|NN .|. 
Free|NNP VO|NNP 2|CD +|NN binds|NNS to|TO sCD39|NN through|IN two|CD hydroxyl|NN groups|NNS and|CC two|CD water|NN molecules|NNS that|WDT are|VBP hydrogen|NN bonded|VBN to|TO other|JJ residues|NNS of|IN sCD39|NN .|. 
Once|RB free|JJ VO|NNP 2|CD +|NN occupies|VBZ the|DT catalytic|JJ site|NN ,|, the|DT enzyme|NN has|VBZ to|TO either|CC release|VB the|DT metal|NN or|CC correct|VB the|DT conformation|NN before|IN the|DT substrates|NNS are|VBP recruited|VBN properly|RB .|. 

Conclusions|NNP VO|NNP 2|CD +|NN can|MD functionally|RB substitute|VB for|IN Ca|MD 2|CD +|NN as|IN a|DT cofactor|NN for|IN sCD39|NN .|. 
Four|CD different|JJ EPR|NNP spectra|NN are|VBP obtained|VBN for|IN VO|NNP 2|CD +|NN bound|VBN in|IN the|DT presence|NN of|IN different|JJ nucleotides|NNS and|CC in|IN the|DT absence|NN of|IN nucleotide|NN .|. 
The|DT protein|NN ligands|NNS for|IN VO|NNP +|NN 2|CD in|IN the|DT presence|NN of|IN ATP|NNP are|VBP suggested|VBN to|TO be|VB carboxyl|NN and|CC amino|JJ groups|NNS ,|, while|IN those|DT in|IN the|DT presence|NN of|IN ADP|NNP are|VBP probably|RB carboxyl|NN and|CC hydroxyl|NN groups|NNS .|. 
The|DT mechanism|NN of|IN sCD|NN catalysis|NNS is|VBZ discussed|VBN .|. 
These|DT results|NNS will|MD provide|VB guides|NNS for|IN further|JJ studies|NNS of|IN the|DT catalytic|JJ mechanism|NN of|IN NTPDases|NNP .|. 

Materials|NNS and|CC Methods|NNP Reagents|NNP ATP|NNP ,|, ADP|NNP ,|, ADPNP|NNP ,|, AMPCP|NNP were|VBD purchased|VBN from|IN Sigma|NNP (|( St.|NN 
Louis|NNP ,|, MO|NNP )|) .|. 
Zeocin|NNP ,|, High-|NNP Five|CD medium|NN were|VBD purchased|VBN from|IN Invitrogen|NNP (|( Carlsbad|NNP ,|, CA|NNP )|) .|. 

Cell|NNP culture|NN and|CC preparation|NN of|IN soluble|JJ CD39|NN sCD39|NN transfected|JJ stable|JJ HighFive|NNP ™|NN insect|NN cells|NNS were|VBD cultured|JJ as|IN described|VBN by|IN Chen|NNP and|CC Guidotti|NNP [|NN 25|CD ]|NN .|. 
Soluble|NNP CD39|NN were|VBD purified|JJ as|IN described|VBN [|NN 25|CD ]|NN with|IN some|DT modifications|NNS .|. 
After|IN concanavalin|NN A-Sepharose|NNP 4|CD B|NNP and|CC nickel|NN affinity|NN column|NN chromatography|NN ,|, the|DT ammonium|NN sulfate|NN precipitated|VBD sCD39|NN was|VBD collected|VBN and|CC resuspended|JJ in|IN about|IN 50|CD μl|NN of|IN 40|CD mM|NN Tris-|NNP HCl|NNP (|( pH7.5|NN )|) .|. 
This|DT sample|NN was|VBD loaded|VBN on|IN a|DT Superose-12|NN HR|NNP gel|NN filtration|NN column|NN from|IN Pharmacia|NNP Biotech|NNP equilibrated|JJ with|IN 40|CD mM|NN Tris-|NNP HCl|NNP (|( pH7.5|NN )|) .|. 
The|DT fractions|NNS containing|VBG the|DT major|JJ peak|NN were|VBD collected|VBN ,|, and|CC the|DT solvent|JJ was|VBD changed|VBN to|TO 20|CD mM|NN Hepes|NNP (|( pH8.0|NN )|) ,|, 120|CD mM|NN NaCl|NNP ,|, 5|CD mM|NN KCl|NNP with|IN an|DT YM30|NN centricon|NN from|IN Millipore|NNP .|. 
The|DT final|JJ volume|NN of|IN the|DT sample|NN was|VBD around|IN 200|CD μl|NN ,|, and|CC the|DT concentration|NN of|IN sCD39|NN was|VBD around|IN 0.1|CD mM|NN .|. 

Concentrations|NNP of|IN proteins|NNS were|VBD determined|VBN using|VBG D|NNP C|NNP Protein|NNP Assay|NNP from|IN BIO-RAD|NNP using|VBG the|DT provided|VBN protocol|NN .|. 

Nucleotidase|NNP activity|NN assay|NN and|CC nucleotide|NN separation|NN by|IN HPLC|NNP The|DT reactions|NNS were|VBD carried|VBN out|IN in|IN 20|CD mM|NN HEPES-Tris|NNP (|( pH|NN 7.0|CD )|) ,|, 120|CD mM|NN NaCl|NNP ,|, and|CC 5|CD mM|NN KCl|NNP ;|: they|PRP were|VBD started|VBN by|IN adding|VBG nucleotides|NNS at|IN 37|CD °|NN C.|NN 
After|IN incubation|NN for|IN 15|CD minutes|NNS ,|, the|DT reactions|NNS were|VBD stopped|VBN with|IN 2|CD %|NN perchloroacetic|JJ acid|NN 

Nucleotides|NNP were|VBD separated|VBN by|IN HPLC|NNP on|IN an|DT anion|NN exchange|NN column|NN (|( a|DT 10|CD ×|NN 0.46|CD mm|NN SAX|NNP column|NN from|IN Rainin|NNP Instruments|NNP )|) based|VBN on|IN the|DT method|NN of|IN Hartwick|NNP and|CC Brown|NNP [|NN 29|CD ]|NN .|. 
The|DT low|JJ concentration|NN buffer|NN (|( A|DT )|) was|VBD 0.08|CD M|NNP NH|NNP 4|CD H|NNP 2|CD PO|NNP 4|CD (|( pH3.8|NN )|) ,|, and|CC the|DT high|JJ concentration|NN buffer|NN (|( B|NNP )|) was|VBD 0.25|CD M|NNP NH|NNP 4|CD H|NNP 2|CD PO|NNP 4|CD (|( pH4.95|NN )|) with|IN 8|CD mM|NN KCl|NNP .|. The|DT gradient|NN used|VBN was|VBD 4|CD min|NN ,|, 0|CD -|: 2.5|CD %|NN (|( B|NNP )|) ;|: 26|CD min|NN ,|, 2.5|CD -|: 25|CD %|NN (|( B|NNP )|) .|. 
Equilibration|NNP was|VBD done|VBN with|IN buffer|NN (|( A|DT )|) for|IN 10|CD minutes|NNS ,|, and|CC the|DT flow|NN rate|NN was|VBD 1|CD ml|NN /|NN min.|NN 

Preparation|NNP of|IN VO|NNP 2|CD +|NN solution|NN Vanadyl|NNP and|CC nucleotide|NN solution|NN were|VBD prepared|VBN according|VBG to|TO Houseman|NNP et|CC al.|NN [|NN 21|CD ]|NN .|. 
Dissolved|NNP molecular|JJ oxygen|NN was|VBD removed|VBN from|IN solutions|NNS by|IN purging|VBG with|IN dry|JJ nitrogen|NN gas|NN .|. 
Stock|NN vanadyl|NN and|CC nucleotide|NN solution|NN were|VBD thawed|JJ on|IN ice|NN ,|, and|CC mixed|JJ at|IN 1|CD :|: 1|CD molar|NN ratio|NN by|IN vigorous|JJ stirring|VBG .|. 
Then|RB VO|NNP 2|CD +|NN -|: nucleotide|NN complexes|NNS were|VBD added|VBN to|TO purified|JJ sCD39|NN at|IN 1|CD :|: 1|CD molar|NN ratio|NN ,|, mixed|JJ ,|, and|CC incubated|JJ for|IN 5|CD minutes|NNS on|IN ice|NN before|IN they|PRP were|VBD transferred|VBN into|IN EPR|NNP tubes|NNS .|. 
Once|RB the|DT samples|NNS were|VBD in|IN EPR|NNP tubes|NNS ,|, they|PRP were|VBD immediately|RB frozen|VBN in|IN liquid|JJ nitrogen|NN ,|, and|CC stored|VBD in|IN liquid|JJ nitrogen|NN before|IN using|VBG .|. 

EPR|NNP Measurement|NNP CW-EPR|NNP experiments|NNS were|VBD carried|VBN out|RB at|IN X-|NNP band|NN (|( 9|CD GHz|NNP )|) using|VBG a|DT Bruker|NNP 300|CD E|NNP spectrometer|NN with|IN a|DT TE102|NN rectangular|JJ standard|JJ cavity|NN and|CC a|DT liquid|JJ nitrogen|NN flow|NN cryostat|NN operating|VBG at|IN 150|CD K.|NN 
Simulations|NNP of|IN these|DT EPR|NNP spectra|NN were|VBD accomplished|VBN with|IN the|DT computer|NN program|NN QPOWA|NNP [|NN 30|CD 31|CD ]|NN )|) .|. 

To|TO estimate|VB the|DT types|NNS of|IN groups|NNS that|WDT serve|VBP as|IN equatorial|NN ligands|NNS to|TO VO|NNP 2|CD +|NN in|IN each|DT condition|NN ,|, the|DT observed|VBD values|NNS of|IN A|DT ||NN ||NN derived|VBN from|IN simulation|NN of|IN the|DT EPR|NNP spectrum|NN by|IN QPOWA|NNP were|VBD compared|VBN with|IN the|DT coupling|NN constants|NNS obtained|VBN from|IN model|NN studies|NNS [|NN 24|CD 32|CD ]|NN using|VBG :|: A|DT ||NN ||NN calc|NN =|SYM Σ|NN n|NN i|NNP A|DT ||NN ||NN i|NNP /|NN 4|CD where|WRB i|NNP represents|VBZ the|DT different|JJ types|NNS of|IN equatorial|NN ligand|NN donor|NN groups|NNS ,|, n|NN i|NNP (|( =|SYM 1|CD -|: 4|CD )|) is|VBZ the|DT number|NN of|IN ligands|NNS of|IN type|NN i|NNP ,|, and|CC A|DT ||NN ||NN i|NNP is|VBZ the|DT measured|VBN coupling|NN constant|JJ for|IN equatorial|NN donor|NN group|NN i|NNP [|NN 24|CD ]|NN .|. 
Similar|JJ equations|NNS were|VBD used|VBN to|TO calculated|VBN g|SYM ||NN ||NN from|IN a|DT given|VBN set|NN of|IN equatorial|NN ligands|NNS for|IN comparison|NN with|IN those|DT derived|VBN experimentally|RB .|. 

