1. 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

2. 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

3. 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

4. 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

7. 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

8. 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

9. 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

10. 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

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

12. 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

13. 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

14. 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

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

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