trans

174 PROTEIN METABOLISM
the C-terminus. Endopeptidases hydrolyze
internal peptide bonds and are frequently referred
to as proteinases or proteases. The modern
classification of peptidases, as developed
by A.J. Barrett, divides them first by catalytic
type (according to the chemical group mainly
responsible for catalysis), then by molecular
structure, and finally the individual peptidases.
Two major catalytic types have been identified:
those enzymes in which the nucleophilic
attack of the peptide bond is performed by a
group in the protein (the sulfhydryl of cysteine
in the cysteine peptidases, and the hydroxyl of
serine or threonine in the serine or threonine
peptidases), and those enzymes in which the
nucleophile is water, through a mechanism
involving two aspartyl residues (aspartic peptidases)
or a metal ion (metallopeptidases).
The first approach for the classification of
a new peptidase within a catalytic class is
usually based on its pattern of response to
inhibitors, but frequently a definitive classification
requires knowledge of its full amino
acid sequence. Within each of the five catalytic
classes, enzymes are classified by molecular
structure into clans (proteins with a similar
folding pattern) and, within clans, into families
(enzymes with significant sequence homology).
To date, more than 130 peptidase families
have been described, including some
unclassified peptidase families whose reaction
mechanisms are still undetermined.
Peptidases belonging to all the catalytic classes
have been described in parasites. Most of the
enzymes studied so far are proteinases, but in
some cases exopeptidases have been well
characterized.
Proteolytic enzymes of protozoa
Cysteine proteinases
These enzymes are abundant in protozoa and
often seem to be the major proteolytic activities
present. In most cases where the genes encoding
these enzymes have been cloned, they predict
the synthesis of a pre-pro-enzyme, similar
to the precursors of the papain family (C1
according to the Barrett nomenclature); in the
case of trypanosomatids, a number of enzymes
with an additional C-terminal extension have
also been described.
Cysteine proteinases are particularly abundant
in amastigotes of Leishmania mexicana,
in epimastigotes of T. cruzi, and in bloodstream
trypomastigotes of T. brucei. Coombs and
co-workers have classified these enzymes into
three types: Types I and II, both cathepsin L-like,
and Type III, cathepsin B-like. The genes encoding
Type I cysteine proteinases predict a long
C-terminal extension, are present in multiple
copies, are arrayed in tandem, and frequently
several genes are simultaneously expressed,
generating a mixture of isoforms. The expression
of Type I genes is developmentally regulated
at different levels in different stages of the
parasite life cycle. Type I enzymes have been
found in all Trypanosomatids studied to date,
including the Leishmania, T. cruzi (cruzipain,
also known as GP57/51 or cruzain), T. brucei
(trypanopain), Trypanosoma congolense (congopain),
Trypanosoma rangeli, and Crithidia
fasciculata. In the case of the Type I cysteine
proteinases of L. mexicana (CPb) and of
cruzipain, a complete tandem array has been
sequenced; the gene at the 3 end, which has a
shorter C-terminal extension, is not expressed.
Type I cysteine proteinases (CPb in L. mexicana)
are cathepsin L-like endoproteinases
with good gelatinolytic activity. The C-terminal
extension, which has substantial homology
among different trypanosomatids, and contains
eight conserved cysteine residues, is lost
during processing in T. brucei and L. mexicana;
in these cases, the mature proteinases are similar
in size to papain. In contrast, cruzipain,
congopain and the Type I cysteine proteinases
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA