Transcript Enzyme Chemistry, 252600

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
1. Detecting the number of sequence of these intermediates and
processes
2. Defining their approximate nature
3. Measuring the rate constants
4. 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
E + RCO-X
Ks
RCO-X·E
k2
RCO-E
+ XH
k3
RCO2H + E
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
1. The rate-determining breakdown of a common intermediate
implies a common value of Vmax or kcat
2. 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
1.
2.
3.
4.
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