Structural Biology: Chapter 7 — Case studies in mechanism

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7.1 Haemoglobin #

7.1.1 The fold and the haem #

Each globin is helices A–H around ferrous protoporphyrin IX. Four porphyrin nitrogens and the proximal histidine, His F8, coordinate the iron. Oxygen, when bound, is the sixth ligand. The distal histidine, His E7, is not a ligand. It hydrogen-bonds to O2 and hinders the linear geometry carbon monoxide prefers. Bare haem binds CO on the order of 20,000 times more tightly than O2; the pocket reduces that preference to about 200-fold. The residual preference is why CO is still toxic. Myoglobin is a monomer with a hyperbolic curve. Haemoglobin is α2β2. The α1β1 contact stays put; the α1β2 contact slides. α and β are different enough that an effector can sit between the β chains only.

7.1.2 The quaternary shift #

Deoxyhaemoglobin is T; oxyhaemoglobin is R. In deoxy haem the high-spin iron sits about 0.5 Å out of the mean porphyrin plane, towards His F8. Oxygen pulls the low-spin iron into the plane and helix F moves with the histidine. One αβ dimer then rotates about 15° on the other, salt bridges that stabilise T break, and the interface shifts by several ångströms. Perutz’s mechanism is that coupling. Later structures add tertiary intermediates; two states remain the right first model. Oxygen binds more tightly to R, so each ligand pushes the equilibrium towards R. The Hill coefficient is not the number of sites. Human haemoglobin has four sites and a coefficient of about 2.8. A value of 1 would be myoglobin. A value of 4 would be infinite cooperativity. About 2.8 is strong and incomplete, which is what a finite T–R equilibrium produces. The Bohr effect is the same switch read in protons: lower pH stabilises T. His146β is a major contributor because its pKa is higher in T. Gas transport in the organism is Animal and Human Physiology.

7.1.3 2,3-Bisphosphoglycerate #

2,3-Bisphosphoglycerate binds once per tetramer in the central cavity of deoxyhaemoglobin, between the β chains, contacting Val NA1, His 2, Lys 82, and His 143 of both β chains. The R cavity is too narrow, so the effector stabilises T and promotes unloading. Stripped haemoglobin holds oxygen too tightly. Fetal γ replaces His 143 with serine, binds 2,3-BPG less tightly, and therefore takes oxygen from maternal adult haemoglobin. The same T state displays the Val 6 contact of haemoglobin S, which is why sickling is a deoxy event.

7.2 Serine proteases #

7.2.1 The triad and the oxyanion hole #

Chymotrypsin, trypsin, and elastase share a fold. In chymotrypsin numbering the catalytic triad is Ser 195, His 57, and Asp 102. Serine is the nucleophile. Histidine takes its proton during attack on the substrate carbonyl and later protonates the leaving group. Aspartate orients the histidine; the resting proton stays on the histidine, not on the aspartate, despite an older charge-relay drawing. The tetrahedral intermediate puts its negative oxygen in the oxyanion hole, the backbone NH groups of Gly 193 and Ser 195. Collapse leaves an acyl-enzyme on Ser 195; water then attacks, and the acid leaves. Values of kcat and Km are Biochemistry. The zymogen already contains the triad. Cleavage to free Ile 16 lets it salt-bridge to Asp 194 and order the hole and the pocket. Subtilisin places the same three residues on an unrelated fold: convergent evolution, not one ancestry.

7.2.2 The specificity pocket #

Catalysis is shared. The S1 pocket is not.

Editing the pocket changes the substrate without redesigning the triad. Specificity is binding geometry. Rate enhancement is the nucleophile, the base, and the hole.

7.3 The ribosome #

7.3.1 An RNA active site #

A bacterial ribosome is about 2.5 MDa and roughly two-thirds RNA. The 2009 Nobel prize to Yonath, Ramakrishnan, and Steitz recognised the structures of the peptidyl-transferase centre and the decoding site. Noller had already found activity in a large subunit stripped of most protein. The maps settled the argument: no protein comes within about 18 Å of peptide-bond formation. The inner shell is 23S rRNA. Aminoacyl-tRNA occupies the A site, peptidyl-tRNA the P site, and deacylated tRNA the E site. On the small subunit, A1492, A1493, and G530 of 16S rRNA, in Escherichia coli numbering, swing in only when the codon–anticodon pair has Watson–Crick shape. The factor cycle that spends GTP is Molecular Biology. The chemistry is attack by the A-site amino group on the P-site ester, and it requires the 2′-hydroxyl of the P-site tRNA’s terminal adenosine. That is substrate-assisted catalysis in an RNA active site, not a missing protein base. Antibiotics jammed in the tunnel or at the decoding site block a particular step; doses are not the structural point.

7.4 ATP synthase #

7.4.1 Rotary catalysis #

How the proton-motive force is generated, and ATP per proton, are Biochemistry. The structural mechanism is rotation. Boyer’s binding-change idea was that the three sites take turns and that the difficult step is release of tightly bound ATP. Walker’s 1994 structure of F1 showed it in one crystal: alternating α and β subunits around γ, the three β subunits in three different nucleotide states, one empty. Catalysis sits on β. A P-loop, the Walker A motif, binds the phosphates. Because γ contacts each β differently, a 120° turn cycles the sites. A peripheral stalk is the stator, so γ rotates relative to the hexamer rather than spinning the whole enzyme. The membrane c-ring turns with γ. Each c subunit has a carboxylate in the bilayer; protons reach it through offset half-channels in subunit a, and a conserved arginine between those paths prevents a leak that would not turn the ring. The ring passes the hydrophobic core only when that carboxylate is neutral. The mammalian mitochondrial c-ring has eight subunits; other species use other integers. Noji, Yasuda, Yoshida, and Kinosita watched γ of immobilised F1 turn in 120° steps during hydrolysis, later resolved into substeps of about 90° and about 30°. Synthesis is the reverse, driven from F0. The crystal showed why turning cycles three sites. The filament showed that it turns.

A mechanism, in all four cases, is a change between seen states tied to atoms in the map: helix F, a tetrahedral oxygen, an RNA active site, a rotating stalk.

7.5 Key terms, summary, and self-check #

Key terms. Proximal histidine; T state; R state; Hill coefficient; 2,3-bisphosphoglycerate; catalytic triad; oxyanion hole; peptidyl-transferase centre; binding-change mechanism; rotary catalysis.

Summary. Oxygen pulls the haem iron into the plane, moves His F8, and rotates one haemoglobin dimer about 15° on the other. A Hill coefficient of about 2.8 is strong incomplete cooperativity among four sites. 2,3-BPG stabilises T; fetal haemoglobin binds it less tightly because a histidine is serine. Serine proteases share Ser-His-Asp and an oxyanion hole, and differ in the S1 pocket; subtilisin converged on the chemistry. No protein is within about 18 Å of peptide-bond formation. One F1 crystal shows three catalytic states at once, and γ rotates.

Self-check.

1. How does a shift of about half an ångström at the iron become a quaternary change?

2. Why is a Hill coefficient of about 2.8 evidence of cooperativity, and why is 4 a different claim?

3. Where does 2,3-BPG bind, which state does it stabilise, and why does fetal haemoglobin bind it less well?

4. What do Ser 195, His 57, and the oxyanion hole do, and how does elastase differ from trypsin?

5. What places peptide-bond formation on RNA, and what did one F1 crystal show that a single site could not?

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