| Introduction
A number of viral and eucaryotic
proteins required for signal transduction and regulatory functions being
important in cellular growth control, undergo a lipophilic modification
by the enzyme N-myristoyl-transferase (Nmt). The lipid modification refers
to the cotranslational transfer of myristiate from myristoyl-coenzyme A
in amide linkage to the N-terminal glycine residue of the proteins (Figure
1). The usual irreversible modification is absolutely essential for the
biological functions of most of these proteins. Attachment of the myristoyl
residue provides hydrophobicity to influence the partitioning of proteins
to cellular membranes and can serve to promote protein-protein interactions[1,2]. |
 |
| Figure 1: Nmt-Reaction
The necessity of N-myristoyl-proteins
in various pathogenes, e.g. C. neoformans which causes a fungal
infection [3], or in viruses, e.g. HIV-I [4,5], Polio virus [6] and Hepatitis
B virus [7], make Nmt as a suitable target for anti-fungal and anti-viral
agents [1,2]. |
| Development of an continious assay
of Nmt activity based on fluorescence spectroscopy
Inspired of an assay for the enzyme farnesyl:protein transferase by Pompliano et al. [11], we have developed an efficient continuous assay based on fluorescence spectroscopy by using the increase in hydrophobicity about the reaction center of the acceptor substrate after fatty acid transfer. Change from a polar to nonpolar molecular enviroment causes an enhancement of fluorescence and an accompanying shift to lower wavelength emission maximum of certain fluorophores, like dansyl. We designed the acceptor peptide
substrate GSSK(dansyl)SKPK-NH2 (Figure 2), corresponding to the amino-terminus
of the natural substrate p60src
ö a tyrosine kinase [8]: Lysin was dansylated and the octapeptide was synthesized
by using Fmoc-strategy with solid phase methods. |
 |
| Figure 2: Dansylated octapeptide substrate GSSK(dansyl)SKPK-NH2 in enzyme reaction |
The recombinand Nmt from S. cerevisiae, Nmt1p, which shows high sequence homology with the enzymes from mammals, was overexpressed in E. coli JM101 [12] and purified by gel chromatography. Nmt1p was added to a solution of
the dansylated peptide substrate and myristoyl coenzyme A in Tris buffer
(pH7.5) at 30 °C. With excitation at 330 nm the increase of emission
at 495 nm was measured (Figure 3). |
 |
| Figure 3: fluorescence emission spectra with lex = 330 nm and lem = 495 nm |
In the linear range of emission increase the enzyme follows the Michaelis Menten kinetic and the Km for the peptid was determined to be 25 µM. |
| Mechanistic investigations - synthesis
of an nonhydrolysable substrate analogue
Myristoyl-carba(dethia)-coenzyme
A 7 is a nonhydrolysable substrate analogue of myristoyl-coA. The
sulfur atom is replaced by a methylene group. For mechanistic studies of
catalysis with nuclear magnetic resonance spectroscopy the carbonyl C-atom
shall be labeled with 13C-isotope.
Therefore an efficient synthesis has been developed: |
 |
| Figure 4: Synthesis of myristoyl-carba(dethia)-coenzyme A 7 |
The Synthesis was carried out with an over-all yield of 23 % referring to myristic acid 1. |
| References
[1] D.A. Rudnick, C.A. McWherter,
G.W. Gokel, J.I. Gordon, Adv. Enzymol. Relat. Areas Mol. Biol. 1993,
67,
375-430 |