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Methods for Studying Enzymatic Reactions 77
residue bearing a radioactive label. In order to identify this active site residue
the inactivated enzyme is broken down into peptide fragments using a protease
enzyme, and the peptide fragment containing the 14 C label is sequenced. At the
position in the peptide sequence containing the radiolabelled inhibitor a nonstandard
amino acid is found and the radioactive label is released from the
peptide. This method is known as peptide mapping.
The covalent attachment of an inhibitor or substrate to an enzyme can also
be analysed by ‘weighing’ the protein. The technique of electrospray mass
spectrometry can be used to determine accurately the molecular weight of
pure proteins of up to 50 kDa to an accuracy of 1 Da! The molecular weight
of a covalently modiWed enzyme can, therefore, reveal the molecular weight of
the attached small molecule.
Finally, with the advent of modern molecular biology techniques it has
become possible to speciWcally replace individual amino acids in an enzyme by
site-directed mutagenesis. This method involves speciWcally altering the sequence
of the gene encoding the enzyme in such a way that the triplet codon
encoding the amino acid of interest is changed to that of a non-functional amino
acid such as alanine. The mutant enzyme can then be puriWed and tested for
enzymatic activity. In this way the precise role of individual amino acids implicated
by modiWcation studies or sequence alignments can be explored.
The ultimate solution to identifying active site amino acid groups is to solve
the three-dimensional structure of the whole enzyme. This is most commonly
done by X-ray crystallography, which depends on obtaining a high-quality
crystal of pure enzyme that is suitable for X-ray diVraction. Recent developments
in multi-dimensional NMR spectroscopy have allowed the structure
determination of small proteins up to 20 kDa in size. Most of the detailed
examples that we shall meet in later chapters have been analysed both by
X-ray crystallography and by several of the above methods.
Problems
(1) Using the data given in Table 4.2, calculate the turnover number (k cat ) for
the enzyme being puriWed. Assume that the Wnal puriWed enzyme is 100%
pure, and that the enzyme contains one active site per monomer. The
subunit molecular weight is 28 kDa.
(2) From the data below obtained from the rate of an enzyme-catalysed reaction
at a range of substrate concentrations, calculate the K M and v max of the
enzyme for this substrate. If 1:65 mg of enzyme was used for each assay, and
if the molecular weight of the enzyme is 36 000, work out the turnover
number for the enzyme for this substrate. Hence calculate the catalytic
eYciency (k cat =K M ) for this substrate.