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PROTEINS: Structure, Function, and Genetics 28:461–462 (1997)
Subtilisin Enzymes: Practical Protein
Engineering, edited by Richard Bott and
Christian Betzel. New York: Plenum Press,
1996, $79.50.
Members of the subtilisin protein family are serine
proteinases that have been the subjects of study by
many laboratories for the past several decades. A
primary reason for this intense interest is because of
the commercial use of these enzymes. Thus, as the
tools for protein engineering are emerging, they are
being applied to subtilisin to change its properties to
enhance the commercial potential of this enzyme. In
1992, in Hamburg, Germany, the first International
Symposium on Subtilisin Enzymes was held, bringing together representatives of most laboratories in
the world that were studying subtilisins. Subtilisin
Enzymes is the proceedings from this unique meeting. Despite the fact that the articles are now dated,
this compendium is very worthwhile reading. The
breath of the studies represented in this work is
quite remarkable, but almost without exception, the
focus of the research relates the enzyme structure to
its function or its function to its structure. The work,
as a whole, is a snapshot in time of the early days of
protein engineering.
The book is divided into five sections, each related
to one of the sessions at the meeting. The first section
is entitled Structural Studies of Enzymes and Enzymes in Complex with Inhibitor. Most of the articles
describe the crystal structures of enzyme-inhibitor
complexes that include eglin-C-inhibiting subtilisin
(Hipler et al.), thermitase (Teplyakov et al.), and
mesentericopeptidase (Dauter et al.), SSI (Streptomyces subtilisin inhibitor) bound to subtilisin (Nonaka
et al.), and the antibiotic bacitracin bound to thermitase and savinase (Pfeffer-Hennig et al.). A highresolution structure (1.95 Å) of eglin C is described
and compared with the structure found in complexes
with subtilisin by Hipler and coworkers. These articles on the complexes contain more than just a
description of the structures. They provide many
ideas and considerations for improving inhibitor
design (i.e., increasing the strength of the interactions). The complex structures are complemented by
a description by Singh and collaborators on efforts to
design subtilisin-specific inhibitors that incorporate
a,b-dehydro residues. In addition, high-resolution
crystal structures of two alkaline enzymes, savinase
and esperase from Bacillus lentus, are described by
Betzel and coworkers.
Two short sections, New Enzymes and StructureFunction Studies, follow. These contain articles on a
hodgepodge of topics with some items of note. A good
summary of the subtilisin-related proteins along
with their sequences and alignment that had been
discovered at the time of the meeting is given by
Siezen. The same author also provides lucid descriptions of his modeling of substrate binding and of
homology modeling efforts. Another modeling study
described by Barnett and Turner describe the use of
the free-energy perturbation techniques to select
mutants that confer thermal stability to subtilisin
BPN. Two biochemical studies are also included that
described the discovery, cloning, and expression of
the enzyme proteinase T from Tritirachium album
(Samal et al.) and that investigate the substrate
binding of Bacillus lentus subtilisin using sitedirected mutagenesis and peptide binding studies
(Gron et al.). In stark contrast with the modeling and
biochemical studies included in these two sections,
an assay is described by Wolff and collaborators that
was designed to measure subtilisin’s performance in
laundry detergents. This actually is a well-written
article that compares the use of subtilisin Carlesburg and BPN, along with engineered variants of
subtilisin BPN.
The fourth section, Biophysical Probes and Mutagenesis, is comprised of reports on studies using
spectroscopic, crystallographic, and biochemical techniques with the tools of molecular biology to characterize subtilisin enzymes and their site-directed
mutants. Classic approaches to investigating the
specificity of Thermoactinomyces vulgaris thermitase using a variety of substrate-analog inhibitors
were described by Peters and coworkers. Enzymatic,
spectroscopic, and crystallographic studies were used
by Müller and Saenger to characterize the inhibition
of proteinase K from Tritirachium album Limber.
Genov and coworkers explored the use of tryptophan
fluorescence as a sensitive probe of protein conformation by examining five different subtilisins. The
influence on stability of the strong calcium binding
site in BPN using site-directed mutagenesis by Gilliland and coworkers and of the weak calcium binding
site in subtilisin Carlesberg random mutagenesis by
Sättler and coworkers are described. In addition,
this section included two presentations on the requirement of the N-terminal prosegment for folding
of the enzymes into a mature active enzyme, given by
Shinde and Inouye for subtilisin BPN and Lee and
Terada for Aqualysin I from Thermus aquaticus
YT-1, respectively.
The final section entitled Protein Engineering of
Subtilisin Enzymes provides articles that illustrate
the state-of-the-art of protein engineering at the
time of the meeting. Studies directed at increasing
the thermal stability of subtilisin BPN are reported
by Oliver and coworkers and subtilisin E from
Bascillus subtilis by Takagi and collaborators. In an
interesting study of savinase, Olsen and coworkers
replace a threonine side chain with others that
displaced water molecules bound within the core of
the protein. Calorimetric studies determined that
these changes destabilize the protein. Reports of
effectively altering the substrate specificity of highalkaline serine proteinase PB92 (van der Laan et
al.), alkaline proteinases of Bascillus lentus (Takagai
et al.), and subtilisin E from Bascillus subtilis are
also presented. Egmond and coworkers described a
systematic study of the effects of site-directed mutants that alter the surface charge of subtilisin and
hence its electrostatic properties. Finally, Bott and
coworkers describe how the proteolytic efficiency of
Bascillus lentus subtilisin was effectively increased
and characterize the resulting changes in structure
by comparing the results of crystallographic struc-
ture determinations. One additional study presented
in this section by Lange and collaborators describes
the use of site-directed mutants to obtain a crystal
structure at pH 10.5, the first structure near the
optimum pH of the enzyme. This was then compared
with a variant structure at pH 6.0.
In summary, this work has much to offer to those
engaged in studies of proteins or in protein engineering. The articles are short and concise, including
many descriptions of what are now considered classic methods in biochemistry, molecular biology, and
protein engineering. The work illustrates the power
of protein engineering even in the early 1990s to
alter properties of subtilisin enzymes for a variety of
reasons and applications: thermal stability, substrate specificity, surface charge, proteolytic efficiency, and calcium-dependent stability.
Gary L. Gilliland
Center for Advanced Research in Biotechnology
of the University of Maryland Biotechnology
Institute and of the National Institute of
Standards and Technology
Rockville, MD
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