156 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
that caused the changes. This mystery was uncov-
ered by the development of biological sciences.
Louis Pasteur claimed the existence and function of
organisms that were responsible for the changes. In
the same era, Justus Liebig observed the digestion of
meat with pepsin, a substance found in stomach flu-
id, and proposed that a whole organism is not neces-
sary for the process of fermentation.
In 1878, Kuhne was the first person to solve the
conflict by introducing the word “enzyme,” which
means “in yeast” in Greek. This word emphasizes
the materials inside or secreted by the organisms to
enhance the fermentation (changes of raw material).
Buchner (1897) performed fermentation by using a
cell-free extract from yeast of significantly high-
light, and these were molecules rather than life-
forms that did the work. In 1905, Harden and Young
found that fermentation was accelerated by the addi-
tion of some small dialyzable molecules to the cell-
free extract. The results indicated that both macromol-
ecular and micromolecular compounds are needed.
However, the nature of enzymes was not known at
that time. A famous biochemist, Willstátter, was
studying peroxidase for its high catalytic efficiency;
since he never got enough samples, even though the
reaction obviously happened, he hesitated (denied)
to conclude that the enzyme was a protein. Finally,
in 1926, Summer prepared crystalline urease from
jack beans, analyzed the properties of the pure com-
pound, and drew the conclusion that enzymes are
proteins. In the following years, crystalline forms of
some proteases were also obtained by Northrop and
others. The results agreed with Summer’s conclu-
sion.
The studies on enzyme behavior were progressing
in parallel. In 1894, Emil Fischer proposed a “lock
and key” theory to describe the specificity and stereo
relationship between an enzyme and its substrate. In
1902, Herri and Brown independently reported a
saturation-type curve for enzyme reactions. They re-
vealed an important concept in which the enzyme-
substrate complex was an obligate intermediate
formed during the enzyme-catalyzed reaction. In
1913, Michaelis and Menten derived an equation
describing quantitatively the saturation-like behav-
ior. At the same time, Monod and others studied the
kinetics of regulatory enzymes and suggested a con-
certed model for the enzyme reaction. In 1959,
Fischer’s hypothesis was slightly modified by Kosh-
land. He proposed an induced-fit theory to describe
the moment the enzyme and substrate are attached,
and suggested a sequential model for the action of
allosteric enzymes.
For the studies on enzyme structure, Sanger and
his colleagues were the first to announce the unveil-
ing of the amino acid sequence of a protein, insulin.
After that, the primary sequences of some hydro-
lases with comparatively small molecular weights,
such as ribonuclease, chymotrypsin, lysozyme, and
others, were defined. Twenty years later, Sanger won
his second Nobel Prize for the establishment of the
chain-termination reaction method for nucleotide
sequencing of DNA. Based on this method, the de-
duction of the primary sequences of enzymes blos-
somed; to date, the primary structures of 55,410
enzymes have been deduced. Combining genetic
engineering technology and modern computerized
X-ray crystallography and/or NMR, about 15,000
proteins, including 9268 proteins and 2324 enzymes,
have been analyzed for their three-dimensional
structures [Protein Data Bank (PDB): http://www.
rcsb.org/pdb]. Computer software was created, and
protein engineering on enzymes with demanded pro-
perties was carried on successfully [Fang and Ford
1998, Igarashi et al. 1999, Pechkova et al. 2003,
Shiau et al. 2003, Swiss-PDBViewer(spdbv): http://
http://www.expasy.ch/spdbv/mainpage.htm, SWISS-
MODELserver: http://www.expasy.org/swissmod/
SWISS-MODEL.htm].FEATURES OF ENZYMES
MOST OF THEENZYMESAREPROTEINSProteins are susceptible to heat, strong acids and
bases, heavy metals, and detergents. They are hydro-
lyzed to amino acids by heating in acidic solution
and by the proteolytic action of enzymes on peptide
bonds. Enzymes give positive results on typical pro-
tein tests, such as the Biuret, Millions, Hopkins-
Cole, and Sakaguchi reactions. X-ray crystallo-
graphic studies revealed that there are peptide bonds
between adjacent amino acid residues in proteins.
The majority of the enzymes fulfill the above criteri-
on; therefore, they are proteins in nature. However,
the catalytic element of some well-known rib-
ozymes is just RNAs in nature (Steitz and Moore
2003, Raj and Liu 2003).