Pyrococcus furiosus is a hyperthermophilic
member of the domain Archaea, one of the three major phylogenetic divisions
of life. There is a paucity of comparative data on the mechanisms of homologous
enzymes from the three domains (Archaea, Bacteria, and Eukarya). Such data
have the potential to enhance our understanding of the evolutionary history
of proteins and organisms, and will certainly contribute to understanding
of enzyme function at high temperatures.
Comparisons of hyperthermostable enzymes with their
counterparts from mesophilic organisms have discovered great similarities
in enzyme mechanisms. The beta-glucosidase of P. furiosus has been compared
to its homologue from the mesophilic bacterium Agrobacterium faecalis
(1). The substrates that are involved in the transition states for these
enzymes are extremely similar, indicating that the mechanism is highly
conserved. Mechanistic and structural studies of the beta-glucosidase (2)
and glutamate dehydrogenase (3, 4) from P. furiosus have demonstrated
the similarities between enzyme homologues from organisms with very different
temperature optima, and that structural data obtained for mesophilic proteins
can be useful for predicting the structure of hyperthermostable proteins.
Recently, the gene for the prolyl oligopeptidase of P. furiosus was cloned
and overexpressed in E. coli as a functional protease (5). The recombinant
prolyl oligopeptidase displayed characteristics identical to those of the
enzyme expressed in P. furiosus (5), affording the opportunity to readily
obtain sufficient quantities of this protein for kinetic studies.
The prolyl oligopeptidase from P. furiosus
(EC 3.4.21.26, formerly termed prolyl endopeptidase) is a serine protease
of unusual specificity, as it cleaves the sequence X-Pro-Y (where X and
Y are any amino acid) on the carboxyl side of proline. Although it possesses
the catalytic triad composed of serine, histidine, and aspartate that is
characteristic of the serine proteases, its primary structure is otherwise
unrelated to that of the chymotrypsin, trypsin, or elastase classes of
serine proteases (6-8). In fact, the order of the amino acids in the primary
structure is different for prolyl oligopeptidase (Ser, Asp, His) than it
is for either the chymotrypsin (His, Asp, Ser) or subtilisin (Asp, His,
Ser) families (9). Prolyl oligopeptidases (POPs) were first isolated from
human tissue (10), and subsequently from the tissues of other mammals (11,
12), fungi (13, 14), bacteria, (15-17) and archaea (5).
In spite of its wide distribution, little is known
about the physiological role(s) of POP in any organism. Nevertheless, the
presence of the enzyme in mammalian brain tissue and its ability to cleave
proline-containing neuroactive peptides (18, 19) have led to the suggestion
that it plays a role in regulation of these peptides. Evidence is
emerging which suggests that POP influences memory in mammals since POP
inhibitors have antiamnesiac properties (20).
As a first step in the detailed investigation of
P. furiosus (Pfu) POP, its substrate specificity, pH activity profiles,
temperature-dependent activity profile, and influences by anions were studied.
In addition, a structural model of Pfu POP was
constructed based on the folding patterns from the POP crystal structure
from Sus scrufa (pig) (21), in which the two enzymes share 32% identity
and 57% similarity at the amino acid level (7). The mammalian (porcine)
and archaeal POPs are compared on the basis of the kinetic studies and
molecular model.
Kinetic and Mechanistic Studies of Prolyl Oligopeptidase from the
Hyperthermophile Pyrococcus furiosus
Accepted for publication in the Journal of Biological Chemistry
2001 (abstract)
Michael N. Harris1, Jeffry D. Madura3,
Li-June Ming*1, Valerie
J. Harwood*2
1 Department of Chemistry and Institute
for Biomolecular Science, University of South Florida, 4202 East Fowler
Avenue, Tampa, FL 33620
2 Department of Biology, University
of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620
3 Department of Chemistry and Biochemistry,
Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282-1530
A Y401--> F401 mutant has been prepared! Kinetic and thermodynamic studies will follow soon!
References