A Suicide Enzyme Substrate

WHY DO THESE TOPICS MATTER ] In The Chemistry of... A Suicide Enzyme Substrate, we shall see how 5-fluorouracil works. Then, at the end of this chapter, we will show how the combination of several of these reactions in series, each setting up the next step like dominos falling in a row, can enable the one-pot preparation of a highly important alkaloid known as tropinone. Tropinone contains the core of several useful pharmaceuticals. 

Conjugate Additions to Activate Drugs 881 A Suicide Enzyme Substrate 883... 

We shall see examples of biochemically relevant conjugate additions in The Chemistry of... Calicheamicin ji Activation for Cleavage of DNA (see Section 19.7B) and in The Chemistry of... A Suicide Enzyme Substrate (Section 19.8). 

SCHEME 11.3 Postulated mechanisms for the inhibition of serine proteases by coumarin derivatives. NuH nucleophile. Pathway a suicide-type inactivation (suicide substrate). Pathway b transient inactivation by formation of a stable acyl-enzyme (alternate substrate-inhibitor). 

The biological activity of a compound can often be affected dramatically by the presence of even a single fluorine substituent that is placed in a particular position within the molecule. There are diverse reasons for this, which have been discussed briefly in the preface and introduction of this book. A few illustrative examples of bioactive compounds containing a single fluorine substituent are given in Fig. 3.1. These include what is probably the first example of enhanced bioactivity due to fluorine substitution, that of the corticosteroid 3-1 below wherein Fried discovered, in 1954, that the enhanced acidity of the fluorohydrin enhanced the activity of the compound.1 Also pictured are the antibacterial (3-fluoro amino acid, FA (3-2), which acts as a suicide substrate enzyme inactivator, and the well-known anti-anthrax drug, CIPRO (3-3). 

Suicide substrates and quiescent affinity labels, unlike the other types of inhibitors discussed in this chapter, form covalent bonds with active site nucleophiles and thereby irreversibly inactivate their target enzymes. A suicide substrate,191 also described by Silverman in a comprehensive review1101 as a mechanism-based inactivator, is a molecule that resembles its target enzyme s true substrate but contains a latent (relatively unreactive) electrophile. When the target enzyme attempts to turn over the... 

An interesting dinically useful prodrug is 5-fluorouracil, which is converted in vivo to 5-fluoro-2 -deoxyuridine 5 -monophosphate, a potent irreversible inactivator of thymidylate synthase It is sometimes charaderized as a dead end inactivator rather than a suicide substrate since no electrophile is unmasked during attempted catalytic turnover. Rathei since a fluorine atom replaces the proton found on the normal substrate enzyme-catalyzed deprotonation at the 5 -position of uracil cannot occur. The enzyme-inactivator covalent addud (analogous to the normal enzyme-substrate covalent intermediate) therefore cannot break down and has reached a dead end (R. R. Rando, Mechanism-Based Enzyme Inadivators , Pharm. Rev. 1984,36,111-142). 

Funaki, T., Takanohashi, Y., Fukazawa, H. and Kuruma, I. (1991) Estimation of kinetic parameters in the inactivation of an enzyme by a suicide substrate. Biochimica et Biophysica Acta, 1078 (1), 43-46. 

Suicide Enzyme Inhibitors. Snicide substrates are irreversible enzyme inhibitors that bind covalently. The reactive anchoring group is catalytically activated by the enzyme itself through the enzyme-inhibitor complex. The enzyme thus produces its own inhibitor from an originally inactive compound, and is perceived to commit suicide. To design a substrate, the catalytic mechanism of the enzyme as well as the nature of the functional gronps at the enzyme active site must be known. Conversely, successful inhibition provides valuable information about the structure and mechanism of an enzyme. Componnds that form carbanions are especially usefnl in this regard. Pyridoxal phosphate-dependent enzymes form such carbanions readily becanse... 

Although detailed structural as well as mechanistic knowledge of an enzyme is desirable, it is by no means necessary in order to design a suicide substrate. This has been shown by Myers and Widlanski (1993) who have designed a simple inhibitor of human prostatic acid phosphatase (PAP), an enzyme that is believed to be involved in the regulation of androgen receptor activity in prostate cells. Since the enzyme shows a preference for hydrolysis of aryl phosphates, the 4-(fluoromethyl)-phenyl phosphate (FMPP) was prepared as a substrate that would, on hydrolysis by the... 

True enough, treatment of PAP with FMPP resulted in a time-dependent inactivation of the enzyme. Competitive inhibitors of PAP protected against inactivation. The authors suggest that FMPP represents a useful basic structure which can be incorporated into the design of more specific phosphatase inhibitors for example, the modified tyrosine 77 could be incorporated into a particular peptide to give a suicide substrate that is selective for a protein phosphatase which preferentially hydrolyses that peptide. 

The preparation of extracts of plant tissue containing active DPOs can be fraught with problems. In the intact plant tissue, both enzyme and substrate are present but are thought to be compartmentalized, with the enzyme bound to membranes and the native substrate ) present in the vacuole. As soon as the tissue is disrupted, these can react together with the very real risk of a suicidal inactivation of the enzyme by its own reaction products. As a result, most isolation procedures for DPOs include additions of ascorbate and/or cysteine to prevent the formation of the reactive quinones. Assuming that the enzyme is... 

As illustrated in Scheme 6.1, once the covalent intermediate is formed, the complex can either follow a normal catalytic cycle or go through a suicide event leading to the irreversible labeling that is necessary for selection. The suicide inhibition efficiency depends on the ratio k /k. This ratio depends on the nature of the suicide substrate and of the enzyme. Therefore, a large excess of suicide substrate as compared to the displayed enzyme is recommended for selection experiments. 

However, when an irreversible inhibitor binds to the enzyme, it cannot be displaced by the substrate and thus binds irreversibly (Fig. 2—51). The irreversible type of enzyme inhibitor is sometimes called a suicide inhibitor because it covalently and irreversibly binds to the enzyme protein, permanently inhibiting it and therefore essentially killing the enzyme by making it nonfunctional forever (Fig. 2—51). Enzyme activity in this case is only restored when new enzyme molecules are synthesized. 

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