DEHYDROGENASES. Dog$ish (Squalus acanthius) Lactate Dehydrogenase (LDH)

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1 Prof. Alejandro Hochkoeppler Department of Pharmaceutical Sciences and Biotechnology University of Bologna

2 Dog$ish (Squalus acanthius) Lactate Dehydrogenase (LDH) Homotetramer, 36.7 kda (monomer), pi 7.18 PDB 6LDH MATLKDKLIGHLATSQEPRSYNKITVVGVGAVGMAAISILMKDLADEVALVDVMEDKLKGEMMDLQHGSLFLHTAKI VSGKDYSVSAGSKLVVITAGARQQEGESRLNLVQRNVNIFKFIIPNIVKHSPDIILVVSNPVDVLTYVAWKLSGLPMHRII GSGNLDSARFRYLMGERLGVHSSHGWVIGEHGDSVPSVWSGMNVASIXXXKLHPLDGTNKDKQDWKKLHKDV VDSAYEVIKLKGYTSWAIGLSVADLAETIMKNLRVHPVSTMVKDFYGIKDNVFLSLPVLNDHGISNIVKMKLKPNEE QQLQKSATTLWDIQKDLKF UniProt P00341 (LDHA-SQUA) MATLKDKLIGHLATSQEPRSYNKITVVGVGAVGMAAISILMKDLADEVALVDVMEDKLKGEMMDLQHGSLFLHTAKI VSGKDYSVSAGSKLVVITAGARQQEGESRLNLVQRNVNIFKFIIPDIVKHSPDIILVVSNPVDVLTYVAWKLSGLPMHRII GSGNLDSARFRYLMGERLGVHSSSHGWVIGEHGDSSVPVWSGMNVAGVSLKELHPELGTDKDKENWKKLHKDV VDSAYEVIKLKGYTSWAIGLSVADLAETIMKNLRVHPVSTMVKDFYGIKNDVFLSLPVLDNHGISNIVKMKLKPDEE QQLQKSATTLWDIQKDLKF

3 Dog$ish (Squalus acanthius) Lactate Dehydrogenase (LDH), 6LDH Homotetramer, 36.7 kda (monomer), pi 7.18 MATLKDKLIGHLATSQEPRSYNKITVVGVGAVGMAAISILMKDLADEVALVDVMEDKLK GEMMDLQHGSLFLHTAKIVSGKDYSVSAGSKLVVITAGARQQEGESRLNLVQRNVNIFKFI IPDIVKHSPDIILVVSNPVDVLTYVAWKLSGLPMHRIIGSGNLDSARFRYLMGERLGVHS SHGWVIGEHGDSVPSVWSGMNVASIKLHPLDGTDKDKQDWKKLHKDVVDSAYEVIK LKGYTSWAIGLSVADLAETIMKNLRVHPVSTMVKDFYGIKDNVFLSLPVLNDHGISNIV KMKLKPNEEQQLQKSATTLWDIQKDLKF α-helix β-strand Loops & Turns AGARQQEGE: open or closed conformation Pyruvate + β- NADH+ H + Lactate + β- NAD +

4 Does LDH feature an ordered mechanism? E + S 1 ES 1 + S 2 ES 1 S 2 EP 1 P 2 EP 1 + P 2 E + P 1 E + S 2 ES 2 + S 1 ES 2 S 1 EP 2 P 1 EP 2 + P 1 E + P 2 E + S 2 ES 2 E + S 2 + S 1 + S 1 ES 1 ES 1 Products Products hance & Neilands, 1952, J. Biol hem., 199, 383. ε of β- NADH = 6200 M - 1 cm μm β- NADH Abs = 0.43 LDH shifts the λ max of β- NADH to 330 nm Δε = 2000 M - 1 cm - 1 K D of LDH- β- NADH (5 ): 7 μm?

5 LDH from beef heart muscle. Binding of β- NAD, pyruvate, or lactate. [LDH] constant, [S] variable. Pi buffer, ph 6.8. Ultracentrifugation ( g, h). [LDH] t (µm) [β-nad] t (µm) [ES n ] (µm) [β-nad] f (µm [LDH] t (µm) [β-nad] t (µm) [ES n ] (µm) [β-nad] f (µm Takenaka & Schwert, 1956, J. Biol. hem., 223:

6 LDH from beef heart muscle. Binding of β- NAD, pyruvate, or lactate. [LDH] constant, [S] variable. Pi buffer, ph 6.8. Ultracentrifugation ( g, h). [LDH] t (µm) [Pyruvate] t (µm) [Pyruvate] f (µm) [LDH] t (µm [Lactate] t (µm) [Lactate] f (µm) Takenaka & Schwert, 1956, J. Biol. hem., 223:

7 DEHYDRGENASES Takenaka & Schwert, 1956, J. Biol. hem., 223: k1 E + S ES k -1 Data expressed as [ES] as a function of [S] (β- NAD) [ES] exceeds [Et] (67.5 μm). Multiple binding sites. LDH: oligomer? At equilibrium : k1 [E][S] = k 2 [ES] ([Et ] [ES])[S] = KD [ES] [Et ] K D + [S] = [ES] [S] [E][S] k 1 = = KD [ES] k1 [Et ] K 1= D [ES] [S] [ES] = [Et ][S] K D + [S]

8 Takenaka & Schwert, 1956, J. Biol. hem., 223: k1 E + S k -1 ESn At equilibrium : Data expressed as [ES]/[E t ] as a function of [S] (β- NAD) n = 5 ± 0.5 K D = 850 ± 140 μm LDH is a tetramer (homo) k 1 [E][S] = k 1 [ES n ] [E][S] [ES n ] = k 1 k 1 = K D ([E t ] [ES n ])[S] [ES n ] = K D [E t ] [ES n ] = K D [S] +1 [ES n ] [E t ] = [S] [S]+ K D [ES n ] [E t ] = n[s] [S]+ K D

9 Fraction Bound : [ES n ] [E t ] = [S] [S]+ K D F.B. [S] = 1 [S]+ K D [S] F.B. = K D + [S] Scatchard Plot y = F.B. x= F.B./[S] y = a + bx a = n b = K D Plot of the data by Takenaka & Schwert n = 3.8 (3.6), K D = 0.44 (0.39) mm 1 K D = [S] F.B. 1 K D [S] = 1 F.B. 1 F.B. [S] = 1 F.B. K D F.B. = 1 K D F.B. [S] F.B. = n K D F.B. [S]

10 αa βa αb βb α β βd αd/e βe α1f βf α2f βg βh βj α1g α2g α3g βk βl βm αh A $lexible loop connects sheet βd and Helix αd/e: from A97 to F120 The binding of β- NADH triggers loop movement and active site closure Apoenzyme: open form. Holoenzyme (ternary): closed form.

11 Dog$ish LDH: α/β protein β- sheets sandwiched by α- helices 3 β- sheets located in 2 domains β- NAD(H)- binding & catalytic domain The β- sheets in the catalytic domain are highly distorted.

12 β- NAD(H)- binding domain: βa- βf, αb- α1f, and α3g atalytic domain: βg- βm, α2f- α2g, and αh

13 The conformation of both domains is affected by β- NAD(H) binding

14 Structural Element Deformation (Å/atom) Translation (Å) Rotation ( ) lass αd Major Mover αe Minor Mover α1f Minor Mover α1g Minor Mover α2g Minor Mover αh Major Mover β- sheets (1-3) Static Loop Major Mover βg- βh Minor Mover βj- α1g Major Mover α1g- α2g Minor Mover βk- βl Minor Mover Gerstein & hothia, 1991, J. Mol. Biol., 220:

15 αh αd Green: Major Movers α1g αe Grey: Minor Movers Both structures (Apo & Holo) are shown in Magenta αd: 22 rotation αh: 7 rotation Loop: 35 rotation Loop translates 5.6 Å

16 Apoenzyme Holoenzyme

17 What residues/regions are responsible for the $lexibility of the αd/loop? Analysis of the region A97- F120: Gerstein & hothia, 1991, J. Mol. Biol. 220: alculation of Δ (Å/atom), for all the contiguous sub- regions in A97- F120 Identi$ication of hinges Δ1 0.2 Δ2 0.4 Δ3 1.4 Δ4 1.6 Δ2- Δ3: increase of slope

18 ϕ97: from -105 to -69 ϕ98: from -174 to -146 ϕ106: from -94 to -75

19 hanging the values of the 3 ϕ angles (97, 98, and 106) accounts for the majority of loop movements. Transition from open (apo) to closed (holo) conformation. What interactions do change in the transition from the open to the closed form?

20 H- bonds in the apo (open) and in the holo (closed) loop conformation Transverse H- bonds: N114 E105 G104 A99- N114 A99- N114 Q101- E105 (not shown) Q102- E105 Q102- E105 A99 Q102 These H- bonds are present in the apo and holo forms. Longitudinal H- bonds?

21 N109 S106 Longitudinal H- bonds pen conformation N116 Q112 L110 N114 E105 Longitudinal H- bonds losed conformation R113 N109 Q112 2 H- bonds lost upon the transition from open to closed conformation. How many interactions between the closed form and β- NADH? N116 R113 E105

22 β- NAD(H)- binding: βαβ motif From K23 to V54 D53 V32 KITVVGVGAVGMAAISILMKDLAD- EVALVDV KITVVGVGAVGMAAISILMKDLAD- EVALVDV X: hydrophobic amino acid KITVVGVGAVGMAAISILMKDLAD- EVALVDV GXGXXG: conserved glycines (purple) KITVVGVGAVGMAAISILMKDLAD- EVALVDV D: aspartate or glutamate D53, 2 - H and 3 - H: H- bonds

23 V54 M55 V29 An hydrophobic crevice hosts the Adenine moiety of β- NAD(H). Hydrophobic interactions in the βαβ motif: I24- V49 (5.5 Å, β- β) V26- L51 (5.6 Å, β- β) I24- I49 (6.1 Å, β- β) V52 V27 R100 interacts with PPi (3 Å) R100

24 The active site of dog$ish LDH Distances: H194 R107 D167 H194- D Å xam- R Å xam- H Å 2 xam- βnad 3.2Å xam- R Å xam- R Å β- NAD XAM R170 R107 belongs to the closed loop (Holo)

25 ARG 170 Type A and type B dehydrogenases H 2 NH NH HN H 2 ARG 107 LDH, ADH: type A (H1 is transferred) GAPDH: type B (H2 is transferred) H 2 N H 2 N H 2 N 2 H 1 H - H 3 HN + NH H 2 HIS 194 N R N H - H 2 ASP 167

26 ARG 170 ARG 170 H 2 N H 2 NH H 2 N - NH H H H 3 H 2 N HN + HN NH H 2 H 2 HIS ARG H 2 N H 2 NH H 2 N - NH H H 3 H H 2 N N HN NH H 2 H 2 HIS ARG N R N H - + N R N H - H 2 H 2 ASP 167 ASP 167

27 Bacillus stearothermophilus LDH variants larke et al., 1988, Biochemistry, 27: Binding of pyruvate: Enzyme WT D to N D to A ARG170 H 2 NH NH H 2 HN ARG107 K m (Pyr, mm) k cat (s - 1 ) Binding of lactate and NAD(H): H 2 N H H 2 N - H H 3 H 2 N HN + NH H 2 HIS194 Enzyme WT D to N D to A K m (Lac, mm) N R N H - k cat (s - 1 ) Binding of NAD(H): slightly affected. pka His: not affected (7.1 ± 0.2). H 2 ASP167 H194 assists the binding of pyruvate

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