Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

Abstract

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 10(10)-fold: but how? Our structures of the yeast PPase product complex at 1.15 A and fluoride-inhibited complex at 1.9 A visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (O(nu)) directly attacks PPi with a pK(a) vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and O(nu), in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.

Scheme 1

Figure 1

Three superimposed A-active-site structures. The PPase:FPPi structure is in gray, the PPase:Pi₂^down structure is in yellow and the PPase:Pi₂^up structure is in orange, showing three separate conformations for the PPi/Pi atoms. All H₂O except the nucleophile O_nu are omitted for clarity. The figure shows the similarities between substrate and product binding; the P2^up conformation mimics substrate binding, but hydrolyzed P1 moves away. The inline direction from F or O_nu toward P2 is always preserved.

Figure 2

Geometry in A-PPase active sites. Coordination in: (a) F-PPi; (b) P1; (c) P2^down; (d) P2^up. Key distances discussed in the text are shown. Atoms are colored by B-factors: dark blue, < 5Ų; light blue, 5–8 Ų; purple, 8–11 Ų; magenta, 11–15 Ų; red, >15 Ų. Hydrogen bonds are color-coded by length, with red for potential LBHBs. P2(O2) in c and d is believed to remain coordinated to M1 and M4; the labeling of the other oxygens in P2^down and P2^up is arbitrary. Metal coordination is shown in green and a gray line shows a distance, but without coordination or H-bonding.

Figure 3

Nucleophile generation. Residues in A-PPase:Pi₂ are color coded by the conformation in which they occur. Red, up conformation; yellow, down conformation; gray, in both conformations. Metal coordination is shown with solid and H-bonding with dotted lines. As shown, the backbone ¹¹⁶C⩵O pushes the D117 CO toward O_nu.

Figure 4

Angles around F/O_nu/P2(O4) in A-PPase FPPi, P2^down, and P2^up complexes. The F-P-O angle is consistent with S_n2 attack, but the angles around the O are not close to tetrahedral.