Chap 7. Detection of Intermediates in Enzymatic Reactions The mechanism of an enzymatic reaction defined by: - characterization of all the intermediates, complexes, and conformational states of the enzyme - determination of the rate constants for their interconversion Detecting the number of sequence of these intermediates and processes Defining their approximate nature Measuring the rate constants Searching for the participation of acidic and basic groups

A. Pre-Steady State vs. Steady State Kinetics Pre-steady state kinetics: providing information about the intermediates and an overall reaction pathway in a scientifically acceptable manner Steady state kinetics: the basic weakness: the evidence is always ambiguous. no direct information about the number of intermediates and the minimum number always assumed A combination of the two approaches should be used Once pre-steady state kinetics has given information about the intermediates on the pathway, steady state kinetics becomes much more powerful

Detection of Intermediates: What is “Proof”? Criteria for considering intermediates The intermediate is isolated and characterized The intermediate is formed sufficiently rapidly to be on the reaction pathway The intermediate reacts sufficiently rapidly to be on the reaction pathway

B. Chymotrypsin: Detection of Intermediates by Stopped-Flow Spectrophotometry, Steady State Kinetics, and Product Partitioning Ks k2 k3 E + RCO-X RCO-X·E RCO-E RCO2H + E + XH

1. Detection of Intermediates from a “Burst” of Product Release Initial burst behavior of the reaction (chymotrysin with excess p-nitrophenyl acetate or p-nitrophenyl ethyl carbonate) The accumulation of intermediates: the acylenzyme Some other examples The enzyme is converted to a less active conformational state on combination with the first mole of substrate The dissociation of the product is rate-determining There is severe product inhibition

2. Proof of Formation of an Intermediate from Pre-Steady State Kinetics under Single-Turnover Conditions The strategy Measuring the rate constants k2 and k3 Showing each of these is either greater than or equal to the value of kcat Choosing a substrate, reaction conditions, and finding an assay

Measurement of the rate constant for acylation, k2 Mixing an excess of ester substrate with the enzyme: The acylenzyme is formed, accumulated, and remain constant Three chromophoric procedures Chromophoric leaving group Chromophoric acyl group Chromophoric inhibitor displacement

Measurement of the rate constant for deacylation, k3 The thematic approach Mixing a substrate with an excess or stoichiometric amount of the enzyme: The acylenzyme is formed, consumes all substrates, and is followed by slow hydrolysis Nonthematic methods Forming stable acylenzymes

Characterization of the intermediate X-ray diffraction studies with the acylenzyme Proving acylenzyme exist Further intermediate possible

3. Detection of the Acylenzyme in the Hydrolysis of Esters by Steady State Kinetics and Partitioning Experiments Detection of intermediates by steady state kinetics depending on: The accumulation of an intermediate that is able to react either with an acceptor whose concentration may be varied, or, preferably, with several different acceptors The generation of a common intermediate E-R by a series of different substrates all containing the structure R. This intermediate must be able to react with different acceptors The rate-determining breakdown of a common intermediate implies a common value of Vmax or kcat Partitioning of the intermediate between competing acceptors

4. Detection of the Acylenzyme in the hydrolysis of Amides and Peptides No direct detection of the acylenzyme in the hydrolysis of amides: the acylenzyme does not accumulate The direct detection of the acylenzyme in ester substrates available: Providing a rigorous proof of the acylenzyme with amides

5. The Validity of Partitioning Experiments and Some Possible Experimental Errors To provide a satisfactory proof of the presence of an intermediate, partitioning experiments must be linked with rate measurements Possible errors: the enzymes misbehavior the errors of product ratios caused by indirect analysis the nature of the leaving group affecting the partition ratio nonspecific binding causing an error

C. Further Examples of Detection of Intermediates Alkaline phosphatase Acid phophatase b-Galactosidase Aminoacyl-tRNA synthetases

E. Detection of Conformational Changes Requirement A systematic analysis of the relaxation times on the binding of substrates or analogues Independent corroborative evidence