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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
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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.
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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])
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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.
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UPPs synthesizes lipid carrier for bacterial cell wall assembly Dolichyl pyrophosphate synthase catalyzes the lipid carrier for Glycoprotein syntehsis lipid I
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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?
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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.
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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
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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
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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
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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 ( ▲ )
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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
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Fluorescent probe for ligand interaction and inhibitor binding using stopped-flow E E-Pexcess probe
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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
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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
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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.
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Binding order and conformational change using fluorescence detection
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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
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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
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FPP bound crystal structure Chang, S. Y. (2004) Protein Sci. 971-978 FPP
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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+
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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.
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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
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Both insertion and deletion mutants adopt open form S83(Ala) 5 V82S83 (A) Wild type (B) S83(Ala)5 (C) V82S83
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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
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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
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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
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[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
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Proposed reaction mechanism of UPPs reaction Mg 2+ -IPP in D26 Mg 2+ -D26-FPP Condensation reaction occurs Mg 2+ -PPi out
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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.
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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
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LPS detection kit
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Defense mechanism in horseshoe crab Lectins : biosensors, immobilize and help killing invaders
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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)
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Sequence alignment for TPL-1 N glycosylation site Chen et al., J. Biol. Chem. (2001)
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Sequence alignment for TPL-2 N glycosylation site
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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)
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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
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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)
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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
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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).
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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
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MIC for TPL-1 and TPL-2 in presence of 1mM DTT ● : without DTT ■ : with 1mM DTT ● : without DTT ■ : with 1mM DTT ● ● ● ● ● ● ● ● ● ■ ■ ■ ■ ■ ■■■■■
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LPS induces aggregation of TPL-2 C4S is monomer and C6S is dimer
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Structural model for functional TPL-2
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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
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