1. Pre-steady-state kinetics vs steady-state kinetics 1.The order of binding of substrates and release of product serves to define the reactants present at the active site during catalysis: it does not establish the kinetically preferred order of substrate addition and product release or allow conclusions pertaining to the events occurring between substrate binding and product release. 2.The value of k cat sets a lower limit on each of the first-order rate constants governing the conversion of substrate to product following the initial collision of substrate with enzyme. These include conformational changes in the enzyme- Substrate complex, chemical reactions (including the formation and breakdown of intermediates), and conformational changes that limit the rate of product release. 3.The value of k cat /K M defines the apparent second-order rate constant for substrate binding and sets a lower limit on the second-order rate constant for substrate binding. The term k cat /K M is less than the true rate constant by a factor defined by the kinetic partitioning of the E-S to dissociate or go forward in the reaction. The goal of pre-steady-state kinetics to to establish the complete kinetic pathway Including substrate binding, chemical reaction (substrate through intermediates to product), and product release. E+ SESEXEPE + P k1k1 k2k2 k3k3 k4k4 k-1k-1 k -2 k -3 k -4

2. Fast kinetics Product release step is slow so the steady-state rate = product release rate To measure the rate of chemical step where the product release is much slower, a single-turnover condition needs to be employed. Under single-turnover condition where [E] >[S], product release needs not to be considered. Under multiple-turnover condition where [S] = 4 x [E], a burst kinetics (a fast phase followed by a steady-state phase of product formation) can be observed for a reaction with slower post-chemical step. A special tool Quench-Flow, needs to be used for single-turnover experiment in msec time scale. A Stopped-Flow instrument allows the measurements of ligand interaction and chemical steps.

3. Rapid-Quench fast kinetics instrument Measure the real rate of chemical step (single turnover, [E]>[S]) Measure the product formation burst (multiple turnover, [S] = 4x[E])

4. UPPs (undeca-prenyl pyrophosphate synthase) reaction UPPs catalyzes sequential addition of eight IPP to an FPP molecule, forming an undeca-prenyl pyrophosphate with 55 carbons and newly formed cis double bonds.

5. UPPs synthesizes lipid carrier for bacterial cell wall assembly Dolichyl pyrophosphate synthase catalyzes the lipid carrier for Glycoprotein syntehsis lipid I

6. Steady-state kinetics Enzyme + FPP + [ 14 C]IPP Incubate different periods butanol [ 14 C]products [ 14 C]IPP Counting radiolabel in butanol vs. buffer to determine rate constant kcat = 0.013 s-1 (without Triton) kcat = 2.5 s-1 (with Triton) [ 14 C]IPP [ 14 C]products counting E E E Substrate binding reaction Product release 190-fold increase Reaction or product release is rate limiting?

7. Rapid-Quench fast kinetics instrument Measure the real rate of chemical step under single turnover, ([E]>[S]) EE EE Stop the reaction in msec time scale Rate is not limited by product release.

8. Rapid-Quench fast kinetics to measure the rate constants of IPP condensation Y axis represents the sum of [14C]IPP incorporated 10  M UPPs, 1  M FPP, 50  M [ 14 C]IPP Single turnover experiments Time courses of C20 ( ● ), C25 ( ○ ), C30 ( ■ ), C35 ( □ ), C40 ( ◆ ), C45 ( ◊ ), C50 ( ▲ ), and C55 ( △ ). Pan et al., (2000) Biochemistry 10936-10942

9. evaporate Acidic phosphatase Buffer (pH 4.8) hexane Mobile phase H 2 O:acetone = 1:19 Evaporate to Small volume Product Analysis using TLC PPi OH Reverse phase TLC butanol

10. The rate constants for IPP condensation determined from single-turnover Steady-state kcat is 2.5 s -1 in the presence of 0.1% Triton, which is consistent with IPP condensation rate; and is 190-fold larger than the rate constant (0.013 s -1 ) in the absence of Triton. No Triton

11. EE Multiple turnovers ([S] = 2x[E]) measured by Rapid-Quench EE 0.75  M enzyme, 6  M FPP and 50  M [ 14 C]IPP without Triton The data indicate formation of C 55 ( △ ), C 60 ( ● ), C 65 ( ■ ), C 70 ( ◆ ) and C 75 ( ▲ )

12. UPPs kinetic scheme after product is formed (W/O Triton) So the kcat = 0.013 s-1 In the cells, membrane lipid may play a role of Triton to control the C55 chain length

13. Fluorescent probe for ligand interaction and inhibitor binding using stopped-flow E E-Pexcess probe

14. Synthesis of fluorescent substrate analogue 7-(2,6-dimethyl-8-diphospho-2,6-octadienyloxy)-8-methyl-4- trifluoromethyl-chromen-2-one geranyl pyrophosphate (TFMC-GPP) Chen et al., (2002) J. Am. Chem. Soc. 124, 15217-15224

15. Interaction of the fluorescent probe with UPPs (A) Fluorescence is quenched by UPPs and recovered by replacement with FPP (B) Probe binds to UPPs with 1:1 stoichiometry (A)(B) (C)(D) (C ) Probe binds to UPPs with a k on = 75  M -1 s -1 (D) Probe releases from UPPs (chased by FPP) with a k off = 31 s -1

16. Kinetic scheme for UPPs reaction FPP is released at 30 s -1 UPP is released at 0.5 s -1 The rate constants ~2.5 s -1 for each IPP condensation were derived from Rapid-Quench experiments.

17. Binding order and conformational change using fluorescence detection

18. 3-D structure of E. coli UPPs Two conformers were found: one (closed form) with PEG bound and the other (open form) has empty active site, and the  3-  B loop is invisible Ko et al., (2001) J. Biol. Chem. 47474-47482

19. FPP binding induces conformational change on  3 helix wild-typeW31F has less quench W91F has almost no quench Chen et al., (2002) J. Biol. Chem. 7369-7376

20. FPP bound crystal structure Chang, S. Y. (2004) Protein Sci. 971-978 FPP

21. In UPPs reaction, FPP binding does not require Mg 2+ and IPP binding needs Mg 2+ (also FPP binds first) FPP (or FsPP) quenches the UPPs Intrinsic fluorescence even in the absence of Mg 2+ +Mg 2+ Mg 2+ is required fro IPP binding No Mg 2+

22. UPPs conformational change during catalysis Chen et al., (2002) J. Biol. Chem. 7369-7376 E-FPP -> <- IPP (Mg 2+ ) 10 sec 200 msec E-FsPP -> <- IPP (Mg 2+ ) IPP k on = 2 uM -1 s -1 Reach the top at 2 sec when the product is formed E + FPP is too fast to be observed.

23. Mutations on some residues of the flexible loop (71-83) or change the loop length affect UPPs activity Ko et al., (2001) J. Biol. Chem. 277, 47474-47482 Chang et al., (2003) Biochemistry 42, 14452-9

24. Both insertion and deletion mutants adopt open form S83(Ala) 5  V82S83 (A) Wild type (B) S83(Ala)5 (C)  V82S83

25. UPPs in complex with sulfates, and two Triton Chang et al., (2003) J. Biol. Chem. 278, 29298-29307 Two Tritons C 55 (8 cis) Open form Arg30 Arg39 Arg194 Arg200

26. Co-crystal structures of UPPs complexed with FsPP and Mg 2+ IPP (closed form) Guo et al., (2005) J. Biol. Chem Conformational changes In the active site, Mg 2+ is bound with PPi of FPP

27. Two positions of Mg 2+ in several crystal structures S1 and S2 are SO 4 ions which solved from previous studies. Yellow: UPPs bound with FPP only D26A-IPPMg-IPP-FsPP

28. [Mg 2+ ] dependence of enzyme activity K i = 1 mM +Mg 2+ (1 mM), IPP binding w/o or w 50 mM Mg 2+, no IPP binding

29. Proposed reaction mechanism of UPPs reaction Mg 2+ -IPP in D26 Mg 2+ -D26-FPP Condensation reaction occurs Mg 2+ -PPi out

30. How to measure protein-ligand interaction? 1.Measure k on, k off and K d = k off / k on using the instruments such as stopped-flow and BIAcore etc. 2.Measure the K d using titration (fluorescence titration, isothermal titration calorimetry etc). 3.ELISA assays to measure the MIC (minimum inhibitory concentration). 4.Measure the protein oligomerization status and the K d using AUC (analytic ultra-centrifuge). 5.Km from Michaelis-Menten kinetics.’ 6.Equilibrium gel filtration. 7.Equilibrium dialysis.

31. Structural Requirement and Ligand Specificity of Tachypleus Plasma Lectins (TPL) for Molecular Recognition in Host Defense Horshoe crab, an arthropod dependent on innate immunity. TPL-1 and TPL-2 were previously Isolated from plasma of horseshoe crab

32. LPS detection kit

33. Defense mechanism in horseshoe crab Lectins : biosensors, immobilize and help killing invaders

34. Previous finding, by Liu et al. LPS-4B resin Horseshoe crab plasma CL-4B resin Tachypleus plasma lectin (TPL-1) TPL-2 Chiou et al., J. Biol. Chem (2000)

35. Sequence alignment for TPL-1 N glycosylation site Chen et al., J. Biol. Chem. (2001)

36. Sequence alignment for TPL-2 N glycosylation site

37. SDS-PAGE of purified wild-type and mutant TPLs reducing Non-reducing Lane 1: MW standards Lane 2: TPL-1 Lane 3: TPL-1-N74D Lane 4: TPL-2 Lane 5: TPL-2-N3D Lane 6: TPL-2-C4S Lane 7: TPL-2-C6S Lane 8: TPL-2-C64S Kuo et al., Biochem J. (2006)

38. ELISA Procedure E. coli Bos-12Serial diluted TPL-1 or -2 Add 1st TPL-1 or -2 Ab and then HRP-linked 2nd Ab Incubate and wash Add HRP substrate Detect OD 450

39. TPL-1-N74D is inactive and TPL-2-N3D is active : TPL-1 : N74D TPL-1 : TPL-2 : N3D TPL-2 Kuo et al., Biochem J (2006)

40. ligands MIC to TPL-1 (mM) MIC to TPL-2 (mM) Glucose>50 Galactose>50 Mannose>50 Fucose>50 Maltose>50 Lactose>50 GlcNAc8.4>50 ManNAc12.3>50 GalNAc18.5>50 NANA32>50 LacNAc14.3>50 Acetic acid>50 L-glutamine>50 N-acetyl-glutamine50>50 N,N-Dimethyl-acetamide>50 N,N-Diacetyl-chitobiose9.3>50 N,N,N,N,N-pentaacetyl- chitopentaose 7.250 Peptidoglycan monomer0.078 ND peptidoglycan

41. BIAcore Measurements TPL-1 bound peptidoglycan unit (digested by LytG) with K D = 8 x 10 -8 M (left panel); and bound muramyl-dipeptide with K D = 2.9 x 10 -7 M (right panel). TPL-2 bound LPS with K D = 6.3 x 10-8 M (not shown).

42. ligandsMIC to TPL-1 (mg/ml) MIC to TPL-2 (mg/ml) Lipid A E. coli F583 (Rd)>1 Ra mutant LPS E. coli EH100 ND>1 Delipidized LPS O128:B8ND0.16 LPS E. coli K-235ND0.3 LPS E.coli 026:B6>10.22 LPS E. coli 055:B4>10.15 LPS E. coli 0111:B4>10.28 Peptidoglycan0.12>1 Mannan>1 Lipotechioic acid>1 Peptidoglycan is target for TPL-1 and LPS is recognized by TPL-2 LPS

43. MIC for TPL-1 and TPL-2 in presence of 1mM DTT ● : without DTT ■ : with 1mM DTT ● : without DTT ■ : with 1mM DTT ● ● ● ● ● ● ● ● ● ■ ■ ■ ■ ■ ■■■■■

44. LPS induces aggregation of TPL-2 C4S is monomer and C6S is dimer

45. Structural model for functional TPL-2

46. Applications of TPLs in detecting bacteria and removing endotoxin 100 bacteria/ml can inhibit binding of TPLs to bacteria TPL-2 can inhibit the growth of gram(-) E. coli TPL-2 can be used to remove endotoxin