Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima

Abstract

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78-2.73 micromol/mg/min in the presence of Cu+ (pH 6, 60 degrees C) and 0.03-0.08 micromol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [gamma-32P]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu.E1 approximately P. In fact, the phosphoenzyme is reduced to much lower steady state levels upon addition of Cu+, due to rapid hydrolytic cleavage. Negligible ATPase turnover is sustained by CopA following deletion of its N-metal binding domain (DeltaNMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [gamma-3)P]ATP by the DeltaNMBD and CXXC mutants, with no effect of Cu+ either on its formation or hydrolytic cleavage. Phosphoenzyme formation (E2P) can also be obtained by utilization of Pi, and this reaction is inhibited by Cu+ (E2 to E1 transition) even in the DeltaNMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in DeltaNMBD and slower in CXXC mutant. We propose that Cu+ binding to the NMBD is required to produce an "active" conformation of CopA, whereby additional Cu+ bound to an alternate (transmembrane transport) site initiates faster cycles including formation of Cu.E1 approximately P, followed by the E1 approximately P to E2-P conformational transition and hydrolytic cleavage of phosphate. An H479Q mutation (analogous to one found in Wilson disease) renders CopA unable to utilize ATP, whereas phosphorylation by Pi is retained.

FIGURE 1.

CopA constructs used in our experiments. Full-length CopA (WT, CXXC mutant, H479Q mutant) consists of 762 residues including a 36-residue extension (MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSR) at the N terminus. The CXXC mutant has two substitutions (C27A and C30A). In the ΔNMBD, 90 residues (¹Met–⁹⁰Glu) were removed and a 10-residue extension (MDHHHHHHLE) was attached to the N terminus. The molecular weights are 83,361 (WT), 83,297 (CXXC mutant), 83,352 (H479Q mutant), 70,922 (ΔNMBD).

FIGURE 2.

Copper dependence of CopA steady state activity. ATPase activity was measured by colorimetric determination of P_i, at pH 6.0 and 60 °C (see “Materials and Methods”). As indicated in the figure, the measurements were obtained either in the presence of 1 mm BCS or in the absence of BCS following addition of incremental concentrations of CuCl₂.

FIGURE 3.

Double-reciprocal plots of SERCA (A) and CopA (B) steady state hydrolytic activity as a function of ATP concentration. A, the reaction was started at 37 °C by addition of various concentrations of ATP to a medium containing 50 mm KCl, 50 mm MOPS, pH 7, 80 mm KCl, 3 mm MgCl₂, 20 μm CaCl₂, 1 μm A12387 (Ca²⁺ ionophore), 1 mm phosphoenolpyruvate, 50 g/ml pyruvate kinase, and 30 μg of SR protein/ml. B, the reaction was started at 40 °C by addition of various concentrations of ATP to a medium containing 50 mm MES/triethanolamine, pH 6.0, 30% glycerol, 2.0 mm sodium azide, 0.5 mm DTT, 10 mm cysteine/Tris, pH 6.0, 5 mm MgCl₂, 0.01% DDM, 25 μg/ml phosphatidylcholine, 40 μm CuCl₂, 1 mm phosphoenolpyruvate, 50 g/ml pyruvate kinase, and 25 μg of reduced CopA protein/ml. Samples were taken at various time intervals for colorimetric determination of P_i, whereby linear plots of steady state velocity were obtained. The unit of velocity is nanomole/mg/min.

FIGURE 4.

Phosphoenzyme formation by utilization of ATP. A, the reaction was started by addition of 25 μm [γ-³²P]ATP to WT CopA in the presence of 1 mm BCS (absence of Cu⁺) (•), with 40 μm CuCl₂ added in conjunction with [γ-³²P]ATP (○), or by addition of [γ-³²P]ATP following a 2-min preincubation with 40 μm Cu²⁺ (▾). Temperature was 60 °C. B, initial velocity of phosphoenzyme formation as a function of ATP concentration. CopA was preincubated for 2 min with 1 mm BCS before addition of [γ-³²P]ATP. Temperature was 40 °C. See “Materials and Methods” for reaction mixtures.

FIGURE 5.

Electrophoretic analysis of Ca²⁺-ATPase (SERCA) and CopA following phosphorylation with [γ-³²P]ATP. The gels were either stained with Coomassie Blue to evidence protein bands, or scanned with a phosphorimager to detect radioactive phosphoenzyme. The reaction with 25 μm [γ-³²P]ATP was allowed to proceed under conditions yielding the highest steady state levels of phosphorylation for each protein. SERCA, 5 s in ice in the presence of 50 mm MOPS, pH 7, 80 mm KCl, 1 mm MgCl₂, and 20 μm CaCl₂ or 1 mm EGTA. CopA, 3 min at 60 °C, in the presence of 50 mm MES/triethanolamine, pH 6.0, 30% glycerol, 0.5 mm DTT, 10 mm cysteine/Tris, pH 6.0, 5 mm MgCl₂, 0.01% DDM, 25 μg/ml phosphatidylcholine, and either 1 mm BCS or 40 μm CuCl₂. See “Materials and Methods” for details.

FIGURE 6.

Pre-steady state behavior of SERCA (A) and CopA (B) upon sequential additions of ATP and Ca²⁺ or Cu⁺·SERCA (0.05 mg/ml) preincubated for 15 min in a medium containing 50 mm MOPS, pH 7, 80 mm KCl, 1 mm MgCl₂, 1 μm A23187 (Ca²⁺ ionophore), and 1 mm EGTA. The reaction was started at 10 °C by the addition of 25 μm [γ-³²P]ATP and, after 10 s, 1.05 mm CaCl₂ was added. CopA (0.1 mg/ml) was preincubated at 37 °C with 0.1 mm BCS for 2 h, in a reaction mixture as described under “Materials and Methods.” The reaction was started at 60 °C by the addition of 25 μm [γ-³²P]ATP and, after 10 s, 0.14 mm CuCl₂ was added. Samples were taken for determination of phosphoenzyme (•) and P_i (○) at the time intervals as indicated. Each time point shown in the graph is the average of three different determinations. Note the P_i burst in concomitance with EP reduction upon addition of copper.

FIGURE 7.

Phosphorylation of ΔNMBD (A) and CXXC(B) by utilization of ATP. The reaction was started by addition of 25 μm [γ-³²P]ATP to ΔNMBD (A) or CXXC(B). The protein was preincubated with either 1 mm BCS (○) or 40 μm CuCl₂ (•). See “Materials and Methods” for reaction mixtures. The temperature was 60 °C.

FIGURE 8.

Decay of phosphoenzyme obtained with ATP. The phosphoenzyme was obtained by a 3-min incubation with 25 μm [γ-³²P]ATP (temperature, 60 °C), using WT (A), ΔNMBD (B), or CXXC (C) CopA, without preincubation with Cu⁺ or BCS. A chase was then started by addition of 1 mm non-radioactive ATP, in conjunction with 1 mm BCS (○) or 40 μm CuCl₂ (•). The decay reaction was acid quenched at various times as indicated.

FIGURE 9.

Phosphorylation of WT CopA with P_i and subsequent decay. A, phosphorylation was started by the addition of 50 μm [³²P]P_i to WT CopA in conjunction with 1 mm BCS (•) or 40 μm CuCl₂ (○), or following a 2-min preincubation with 40 μm CuCl₂ (▾). B, P_i concentration dependence of the phosphoenzyme equilibrium level, in the presence of 1 mm BCS. Incubation was for 5 min at 60 °C. C, decay was started with a 1 mm non-radioactive P_i chase added to [³²P]phosphoenzyme obtained by a 5-min incubation with 50 μm [³²P]P_i in the absence of both BCS and CuCl_2. The chase medium contained either 1 mm BCS (•) or 40 μm CuCl₂ (○). The temperature was 60 °C.

FIGURE 10.

Phosphorylation of ΔNMBD protein with P_i and subsequent decay. A, phosphorylation was started by the addition of 50 μm [³²P]P_i to ΔNMBD in conjunction with either 1 mm BCS (•) or 40 μm CuCl₂ (○), or following a 2-min preincubation with 40 μm CuCl₂ (▾). B, decay was started with a 1 mm non-radioactive P_i chase added to [³²P]phosphoenzyme obtained by a 5-min equilibration with 50 μm [³²P]P_i in the absence of both BCS and CuCl₂. The chase medium contained either 1 mm BCS (•) or 40 μm Cu²⁺ (○). The temperature was 60 °C.

SCHEME 1.

The diagram suggests that, in the absence of Cu⁺, CopA resides in a basal (E_b) conformation that utilizes ATP or P_i at slow rates. Cu⁺ binding to the NMBD induces transition to an active CopA conformation (E_a), whereby additional Cu⁺ binding to an alternate, transmembrane metal binding (TM-MBS) transport site allows faster cycles, including the E1 to E2 conformational transition, faster phosphoenzyme formation and cleavage, and most importantly transition of E_a1 ∼ P·Cu⁺ to E_a2 ∼ P·Cu⁺. Whereas binding and dissociation of the latter Cu⁺ occurs within each fast cycle, CopA activation by Cu⁺ binding to NMBD occurs with a time constant apparently slower than a single cycle of the activated enzyme.

FIGURE 11.

Phosphorylation of CXXC protein with P_i, and subsequent decay. A, phosphorylation was started by the addition of 50 μm [³²P]P_i to the CXXC mutant in the presence of either 1 mm BCS (•) or 40 μm CuCl₂ (○), or following a 2-min preincubation with 40 μm Cu²⁺ (▾). B, decay was started by addition of 1 mm non-radioactive P_i chase to [³²P]phosphoenzyme obtained by a 5-min equilibration with 50 μm [³²P]P_i in the absence of both BCS and CuCl₂. The chase medium contained either 1 mm BCS (•) or 40 μm Cu²⁺ (○). The temperature was 60 °C.

FIGURE 12.

Phosphorylation of H479Q mutant with P_i, and subsequent decay. A, phosphorylation was started by the addition of 50 μm [³²P]P_i to H479Q in conjunction with either 1 mm BCS (•) or 40 μm CuCl₂ (○). B, decay was started with a 1 mm non-radioactive P_i chase added to [³²P]phosphoenzyme obtained by a 5-min equilibration with 50 μm [³²P]P_i in the absence of both BCS and CuCl₂. The chase medium contained either 1 mm BCS (•) or 40 μm CuCl₂ (○). The temperature was 60 °C.

FIGURE 13.

Alignment of sequences involved in the phosphorylation reaction in the haloacid dehalogenase family. From the top, these sequences correspond to Pho-Ser-Pho, phosphoserine phosphatase from Methanococcus jannaschii; CopA from T. maritima; CopA from A. fulgidus; rabbit skeletal muscle sarcoplasmic Ca²⁺-ATPase; and porcine renal Na⁺/K⁺-ATPase.