Table I.

Mass Spectrometric Analysis of Deuterated (D) and Hydrogenated (H) EI^Ntr

	H	D	% Deuteration1
EINtr (phospho-form)
Calculated mass	66,969.82	70,619.83
Measured mass	66,969.5	70,223.8	89
EINtr, Dephosphorylated 4
Calculated mass	66,889.85	70,609.83
Measured mass	66,890.7	70,213.6	89
EINtr (H356A)
Calculated mass	66,823.75	70,473.73
Measured mass	66,823.8	70,039.2	88
Active Site Peptide, P350–362 6
Calculated mass	1,312.5	1,377.5
Measured mass	1,312.5	1,370.0	88

Mass analysis of the active site peptide and the intact proteins was carried out as described in Methods.

¹The % deuteration is expressed as (Measured mass of D-calculated mass of H/calculated mass of D-calculated mass of H).

²calculated mass corresponds to the protein (mass=66,711.6) which is both phosphorylated (adds 80 mass units) at the active site and gluconoylated [12] (adds 178 mass units) at the site of the His tag sequence.

³The calculated mass of D is expressed as the mass of the protein in which all nonexchangeable hydrogens are replaced by deuterium (perdeuterated protein).

⁴Dephosphorylation was accomplished by treatment with pyruvate and MgCl₂ (see methods).

⁵Calculated mass corresponds to the protein (mass=66,711.6) which is gluconoylated (adds 178 mass units).

⁶Calculated mass corresponds to the protein (mass=66,645.7) which is gluconoylated (adds 178 mass units).

⁷The active site peptide sequence is DGAANSHAAIMVR. The histidine residue is the site of phosphorylation.