Download Epoxyalkyl peptide derivatives as active-site

601st MEETING, ABERDEEN
105
Epoxyalkyl peptide derivatives as active-site-directedinhibitors of asparagine
N-glycosyltransferases
ERNST BAUSE
tnstitut f u r Biochemie, Universitat zu Koln, 0-5000 Koln,
Federal Republic of Germany
Synthetic peptides of defined structure represent excellent tools
for the characterization of substrate specificities of asparagine
N-glycosyltransferases (Bause, 1979; Bause & Hettkamp,
1979). By using this model approach, we recently demonstrated
that triplet sequences of the type Asn-Xaa-Thr(Ser,Cys) are
structural requirements for N-glycosylation, as was postulated
some years ago by Marshall (1974). In addition, these studies
revealed that the hydroxy-amino acid in this particular position
of these tripeptides participates actively in the catalytic
mechanism of transglycosylation. This occurs by promoting, in
a kind of proton-relay system, the necessary hydrogen transfer
from the p-amide of asparagine on to a corresponding basic
group at the active site of the transferase (Bause & Legler,
1981).
On the basis of this knowledge of the catalytic mechanism of
asparagine N-glycosyltransferases, we have designed and
synthesized two hexapeptides as potential active-site-directed
inhibitors for this class of enzymes. Both compounds are derived
from the basic sequence Arg-Asn-Gly-Yaa-Ala-Val-OMe, where
Yaa represents epoxyethylglycine (I) and 2,3-epoxypropylglycine (11). Hence, these derivatives represent substrate
analogues that are carrying a chemically reactive group at a
strategically favoured position for a reaction with an amino acid
side chain at the active site. They were prepared by epoxidation
with p-chloroperbenzoic acid from their corresponding olefinic
peptide precursors. The latter compounds were synthesized by
Abbreviation used: OMe, methoxy (methyl ester).
the solid-phase method as described by Erickson & Merrifield
(1976).
Preliminary experiments showed that neither compound I nor
compound I1 was acting as a glycosyl acceptor under standard
assay conditions, which usually generate soluble glycopeptides
(Bause, 1979). However, when the particulate membrane
fraction was pre-incubated with these derivatives in the presence
of detergents, which are required to facilitate accessibility of the
transferases, a time- and concentration-dependent loss of
enzyme activity is observed with peptide I, whereas no inhibiting
effect is produced by derivative 11. Partial protection against
inhibition by peptide I could be obtained, when the preincubation experiments were carried out in the presence of the
standard acceptor peptide Arg-Asn-Gly-Thr-Ala-Val-OMe.
These observations suggest a specific reaction of inhibitor I at the
active site of the transferases. The inhibitory effect exerted by
compound I was less pronounced when the transferases were
pre-incubated with the standard acceptor peptide before the
addition of the inhibitor. As this treatment reduces the
endogenous pool of glycosyl donor molecules, we conclude that
the inactivation occurs only under conditions where glycosyl
transfer is catalysed.
The various experimental data are consistent with a mechanism of inactivation as outlined in Fig. l(a). For clarity, Fig. l ( b )
shows a model of the catalytic mechanism of N-glycosylation as
proposed recently (Bause & Legler, 1981). We assume that,
comparable with the hydroxy group of threonine or serine, the
oxygen atom of the epoxy function is capable of interacting with
the P-amide of asparagine via hydrogen bonding. As a result of
this contact, the corresponding structure might be recognized as
'active conformation' and consequently be bound and glycosylated. The process of glycosylation is, after binding of the
Dol-PP
E-Enzyme
J
-------c2
I /CH,
I
h'~G1ycosyltransferase
b
Dol-PP-OS
(b)
0
Dol-PP
Tr
\c/
!-Enzyme
J
OH
'"rH
------__--- -- 0:
I
HCH
I
I
H
I
0 HC-CH,
I I I
\NAcY\cHAN/cTc/
I
H
d
B
A
d
B-Enzyme
H\
o\c
N-Glycosyltransferase
b
I
/OS
2-
H,.
I
HCH
H
I I I
\N,cec/N,.
I
II
H
O
0
0:
I
I
HC-CH,
Abbreviations: Dol-PP, dolichyl diphosphate; Dol-PP-OS, dolichyl diphosphate oligosaccharide; B-Enzyme, base at the active site of N-glycosyltransferase.
11
I
C\N/ CH
\c/U
CH
L
A
O
1
Fig. 1. Proposed mechanism of inactivation of asparagine N-glycosyltransferases by the
epoxyethylglycine-containinginhibitor peptide ( a ) and catalytic mechanism of N-glycosylation ( b )according to Bause & Legler ( I 981)
VOl.
H
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epoxyalkyl peptide to the enzyme, initiated by the protonation of
the oxiran ring giving rise to an intermediate with high alkylating
potential. This structure is then stabilized, e.g. by a nucleophilic
attack through the catalytically active base, which normally
accepts the Bamide proton during catalysis. The final product of
this reaction sequence is an inactivated enzyme containing the
glycosylated inhibitor covalently attached via the epoxy function- This type of ~%ction mechanism implies that the
N-glycosyltransferase catalyses its own inactivation in a kind of
‘suicide mechanism’. The various data support our present idea
BIOCHEMICAL SOCIETY TRANSACTIONS
of the functional role of the hydroxy amino acid during the
process of N-glycosylation.
Bause, E. (1979) FEBS Lett. 103,296299
Bause, E. He*amp, H. (1979) FEBs Lett. lm 341-344
Bause, E.Bt Legler, G.(1981)Biochem. J . 195, 639-644
Marshall, R. D.(1974) ~i~-~. so,.. symp.
40, 17-26
Erickson, B. W. & Medieid, R. B. (1976) in The Proteins (Neurath,
H. & Hill, R.L.,eds.), vol. 2, pp. 255-562, Academic Press, London
and New York
1983