The catalytic power of magnesium chelatase: a benchmark for the AAA(+) ATPases

Abstract

In the first committed reaction of chlorophyll biosynthesis, magnesium chelatase couples ATP hydrolysis to the thermodynamically unfavorable Mg(2+) insertion into protoporphyrin IX (ΔG°' of circa 25-33 kJ·mol(-1) ). We explored the thermodynamic constraints on magnesium chelatase and demonstrate the effect of nucleotide hydrolysis on both the reaction kinetics and thermodynamics. The enzyme produces a significant rate enhancement (kcat /kuncat of 400 × 10(6) m) and a catalytic rate enhancement, kcat/KmDIXK0.5Mgkuncat, of 30 × 10(15) m(-1) , increasing to 300 × 10(15) m(-1) with the activator protein Gun4. This is the first demonstration of the thermodynamic benefit of ATP hydrolysis in the AAA(+) family.

Keywords: ATP hydrolysis; ATPases associated with various cellular activities (AAA) magnesium protoporphyrin IX; Gun4; chelatase; chlorophyll biosynthesis.

Figure 1

The insertion of Mg²⁺ into the ring of deuteroporphyrin.

Figure 2

Progress curves showing the characteristic rise and fall of MgD_IX during a magnesium chelatase (0.1 μm ChlD, 0.1 μm ChlI, and 0.4 μm ChlH) catalyzed reaction in the presence (open markers) or absence (closed markers) of an ATP‐regenerating system with (squares) or without (circles) 0.4 μm Gun4. All reactions were performed at 34 °C, 50 mm MOPS, 300 mm glycerol, I 0.1, pH 7.9, 1 mm DTT and contained 8 μm D_IX, 10 mm Mg²⁺ and (A) 0.5 mm MgATP ²⁻, (B) 1 mm MgATP ²⁻ or (C) 5 mm MgATP²⁻.

Figure 3

Steady‐state ratios of product to substrate achieved by magnesium chelatase (0.1 μm ChlD, 0.1 μm ChlI, and 0.4 μm ChlH) with (squares) or without (circles) 0.4 μm Gun4 at 34 °C, 50 mm MOPS, 300 mm glycerol, I 0.1, pH 7.9, 1 mm DTT 10 mm Mg²⁺, ATP‐regenerating system (2 mm PEP, 2 U·mL⁻¹ PK) and (A) varying MgATP ²⁻ at 8 μm D_IX, (B) varying D_IX at 5 mm MgATP ²⁻. The lines in (A) are empirical.

Figure 4

Initial rates of the uncatalyzed dechelation of Mg‐Deuteroporphyrin IX at 34 °C, 50 mm MOPS, 300 mm glycerol, I 0.1, pH 7.9, 1 mm DTT. The fitted line has a slope of 31.7 × 10⁻⁶ ± 0.9 × 10⁻⁶ s⁻¹.