Showing papers by "Christopher T. Walsh published in 1980"

Mechanism-based enzyme inactivation using an allyl sulfoxide-allyl sulfenate ester rearrangement

Abstract: 2-Amino-4-chloro-5-(-nitrophenylsulfinyl)pentanoic acid (1) has been synthesized and shown to induce mechanism-based inactivation of two pyridoxal phosphate dependent enzymes: (1 ) cystathionine y-synthetase, which catalyzes a y-replacement reaction in bacterial methionine biosynthesis; and (2) methionine y-lyase, which catalyzes a y-elimination reaction in bacterial methionine breakdown. The inactivations are irreversible and display saturation kinetics. Each enzyme incorporates roughly I mol of tritium per mol of enzyme monomer when inactivated by 2-amin0-4-chlor0-5-(-nitro[~H]phenylsulfiny1)pentanoic acid (la), confirming that the modification of each protein is covalent and stoichiometric. Substoichiometric labeling (0. I2 mol of tritium per mol of enzyme monomer) is given when methionine y-lyase is fully inactivated by 2-amino-4chlor0-5-[~H]-5-p-nitrophenylsulfinyl)pentanoic acid (lb). Both enzymes, inactivated by 1, are susceptible to reactivation by thiols. Inactivated cystathionine y-synthetase recovers 25% of its catalytic activity upon incubation with excess dithiothreitol, while methinonine y-lyase is 100% reactivated by dithiothreitol, mercaptoethanol, and mercaptopropionate. Reactivation generates p-nitrophenylthiolate anion, which forms, in the case of methionine y-lyase, stoichiometrically with enzyme reactivated. Both enzymes are “protected” from inactivation by 1 in the presence of thiols, which simultaneously generates p-nitrophenylthiol. In the presence of dithiothreitol, the protection reaction gives p-nitrophenylthiol production with pseudo-first-order kinetics. 2-Amino-4-chloro-5-(-tolylsulfinyl)pentanoic acid (2) and 2-amino-4-(p-nitrophenylsulfinyl)-5-chloropentanoic acid (3), the reverse regioisomer of 1, have also been prepared and give no evidence of inactivation of either enzyme. The data are taken to indicate a novel form of suicide inactivation (Scheme 11) wherein P-carbanion-assisted y-halide elimination generates an allyl sulfoxide-enzyme-pyridoxal adduct (4) which undergoes spontaneous 2,3-sigmatropic rearrangement to an electrophilic allyl sulfenate ester (5). The latter is then captured by an enzymic nucleophile to give an inactive enzyme 6, which may be a mixed disulfide or, less likely, a sulfenamide.

[39] Preparation, characterization, and coenzymic properties of 5-carba-5-deaza and 1-carba-1-deaza analogs of riboflavin, FMN, and FAD

Abstract: Publisher Summary This chapter discusses the preparation, characterization, and some of the biological and coenzymic properties of the riboflavin analogs, 5-carba-5-deazariboflavin, and 1-carba-l-deazariboflavin at the riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) levels. These two analogs have carbon substituted for nitrogen at either end of the redox-active ethylenediamine linkage of the isoalloxazine ring and have proven quite useful, in complementary ways, as probes of flavoenzyme mechanisms. Phosphorylation of 5-carba-5-deazariboflavin to yield 5-carbadeazaFMN can be accomplished either chemically or enzymically. The enzymic route, although limited in the amount of material that can conveniently be prepared, appears to be the preferred route in view of some unwanted phosphorylation at the 4-position of the ribityl side chain during chemical phosphorylation. Enzymic phosphorylation of 5-carba-deazariboflavin can be accomplished, either by using adenosine triphosphate (ATP):riboflavin-5′-phosphotransferase (EC 2.7.1.26) from rat liver purified through the fourth step in the purification scheme of McCormick or using the Brevibacterium ammoniagenes flavokinase-FAD synthetase complex. An alternate procedure for obtaining 5-carba-deazaFMN involves the cleavage of 5-carba-deazaFAD by phosphodiesterase.

Chapter 22. Scope and Mechanism of Enzymatic Monooxygenation Reactions

Abstract: Publisher Summary This chapter discusses the scope and mechanism of enzymatic monooxygenation reactions. Dioxygen functions as a simple electron acceptor in biological redox reactions. It can also be activated for insertion of one (monooxygenation) or both (dioxygenation) of its atoms into a cosubstrate molecule by a wide variety of enzymes. These enzymic oxygenations are often key metabolic events in assimilatory sequences, such as catecholamine or steroid hormone biosynthesis, in degradative and detoxifying sequences in liver processing of drugs and toxins, and occasionally in toxifying reactions such as precarcinogen activation in alkylnitrosamine or polycyclic hydrocarbon oxygenative metabolism. Since triplet 02 is spin-unpaired and organic cosubstrates are spinpaired, enzymes have evolved mechanisms for selective acceleration of the kinetic sluggishness of hydroxylations in two ways: (1) by using redox active metals such as copper or iron, and (2) by using a conjugated organic cofactor such as flavin or pterin coenzymes. Iron and copper act as direct 0 2 ligands and electron conduits, while the dihydroflavins and tetrahydropterins probably react via radical mechanisms with 0 2 to yield semiquinone and 0 2 -o with subsequent rapid radical recombination to yield flavin hydroperoxides and pterin hydroperoxides as proximal oxygenation agents.