Monoterpene biosynthesis: mechanism and stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate

The conversion of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate by a partially purified cyclase from sweet marjoram (Majorana hortensis) is considered to proceed by the initial ionization and isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound tertiary allylic intermediate to the monocyclic (+)-(4R)-alpha-terpinyl cation. A 1,2-hydride shift and a second cyclization with water capture of the resulting cation complete the reaction sequence. [6-3H, 14C]Geranyl pyrophosphate, coupled with selective chemical degradation of the resulting sabinene hydrate products, was employed to demonstrate the hydride shift, while separate testing of the linalyl pyrophosphate enantiomers confirmed the involvement of the (3R)-antipode in the cyclization and indicated the cyclization of linalyl pyrophosphate to be faster than the coupled isomerization-cyclization of the geranyl substrate. (1R)- and (1S)-[1-3H, 14C]geranyl pyrophosphates, in conjunction with stereoselective degradations of the biosynthetic products to locate the 3H, were exploited to deduce that configuration at C1 of the substrate was retained in the reaction. These findings suggest the isomerization of the geranyl substrate to be a suprafacial process and the cyclization of the (3R)-linalyl intermediate to proceed via the anti,endo-conformation consistent with the stereo-chemistry of other monoterpene cyclizations and with chemical model studies. Sulfonium ion analogs of the presumptive linalyl and alpha-terpinyl cationic intermediates of the isomerization-cyclization sequence were shown to be potent inhibitors of the enzymatic reaction (Ki = 0.3 and 2.8 microM, respectively), and inhibition was synergized by the presence of inorganic pyrophosphate, indicating that the enzyme recognized and bound more tightly to these ion-paired species than to either cationic or anionic partner alone. Additionally, the enzyme was capable of ionizing (solvolyzing) the noncyclizable substrate analogs 6,7-dihydrogeranyl pyrophosphate and 2,3-methanogeranyl pyrophosphate. These results define the overall stereochemistry of the coupled isomerization-cyclization to sabinene hydrate, demonstrate the 1,2-hydride shift, and confirm the electrophilic nature of this enzymatic reaction type.     

Uncompetitive inhibition of monoterpene cyclases by an analog of the substrate geranyl pyrophosphate and inhibition of monoterpene biosynthesis in vivo by an analog of geraniol

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-o l, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K'i values of 3.2 and 4.7 microM, respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.     

Monoterpene cyclases: use of the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple the isomerization step of the coupled isomerization-cyclization reaction

Enzymes from Salvia officinalis capable of catalyzing the isomerization and subsequent cyclization of geranyl pyrophosphate to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate were examined with the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate in an attempt to dissect the cryptic isomerization step from the normally coupled reaction sequence. The analog inhibited the cyclization of geranyl pyrophosphate and was itself catalytically active, affording acyclic terpene olefins and alcohols as products. The enzymatic products generated from 6,7-dihydrogeranyl pyrophosphate qualitatively resembled the solvolysis products of 6,7-dihydrolinalyl pyrophosphate, yet they constituted a far higher proportion of olefins, suggesting that enzymatic product formation occurs in an environment relatively inaccessible to water. Since the normal cyclization of geranyl pyrophosphate is considered to proceed via preliminary isomerization to the bound tertiary intermediate (3R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3R)-6,7-dihydrolinalyl pyrophosphate which is reionized, and because the subsequent cyclizations are precluded, the resulting cation is either deprotonated or captured by water. In divalent metal ion requirement, pH optimum, and other characteristics, the enzymatic transformation of the analog resembles the normal monoterpene cyclase reaction.     

Monoterpene biosynthesis: mechanistic evaluation of the geranyl pyrophosphate:(-)-endo-fenchol cyclase from fennel (Foeniculum vulgare)

Geranyl pyrophosphate:(-)-endo-fenchol cyclase catalyzes the conversion of geranyl pyrophosphate to (-)-endo-fenchol by a process thought to involve the initial isomerization of the substrate to the tertiary allylic isomer, linalyl pyrophosphate, and the subsequent cyclization of this bound intermediate. Studies with 18O-labeled acyclic precursors and H2(18)O, followed by mass spectrometric analysis of the cyclic product, confirmed that water was the sole source of the carbinol oxygen atom of endo-fenchol, thus indicating the participation of the solvent in terminating this presumptive carbocationic reaction. The isomerization component of the normally coupled reaction sequence was demonstrated directly using the substrate analog 2,3-cyclopropylgeranyl pyrosphosphate and by isolating the corresponding homoallylic analog of linalyl pyrophosphate as a major reaction product. The cyclization component of the reaction sequence was effectively dissected using linalyl pyrophosphate as substrate, and both isomerization and cyclization steps were shown to take place at the same active site of the cyclase, an observation consistent with the efficient coupling of these processes. 2-Fluorogeranyl pyrophosphate and 2-fluorolinalyl pyrophosphate were shown to be effective inhibitors of the cyclase, and the electron-withdrawing substituent was shown to greatly suppress the rate of cyclization of these labeled analogs, indicating that both steps of the coupled isomerization-cyclization sequence are initiated by ionization of an allylic pyrophosphate. Additional evidence for the electrophilic nature of the reaction was obtained by demonstrating the ability of the cyclase to solvolyze other substrate analogs which bear an allylic pyrophosphate, and by showing that cyclization was strongly inhibited by sulfonium analogs of presumptive carbocationic intermediates of the reaction sequence, especially in the presence of inorganic pyrophosphate as counterion. In spite of the fact that the fenchol cyclase terminates the cyclization with an external nucleophile (H2O), the primary mechanistic features of this isomerization-cyclization reaction are similar to those catalyzed by other cyclases that terminate the reaction by deprotonation or cation capture by the pyrophosphate moiety of the substrate.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-bornyl pyrophosphate

Enzymes from Salvia officinalis and Tanacetum vulgare leaf epidermis catalyze the conversion of the acyclic precursor geranyl pyrophosphate to the cyclic monoterpenes (+)- and (-)-bornyl pyrophosphate, respectively. The antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H] Linalyl pyrophosphate (3H:14C = 5.22) was tested as a substrate with the cyclases from both sources to determine the configuration of the cyclizing intermediate. This substrate yielded (-)-bornyl pyrophosphate with 3H:14C ratio greater than 31, indicating specific utilization of (+)-(3S)-linalyl pyrophosphate as predicted. With the (+)-bornyl pyrophosphate cyclase, the 3H:14C ratio of the product was about 4.16, indicating a preference for the (-)-(3R)-enantiomer, but the ability also to utilize (+)-(3S)-linalyl pyrophosphate. (3R)- and (3S)-[1Z-3H]Linalyl pyrophosphate were separately compared to the achiral precursors [1-3H] geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. All functional precursors afforded optically pure (-)-(1S,4S)-bornyl pyrophosphate with the T. vulgare-derived cyclase (as determined by chromatographic separation of diastereomeric ketals of the derived ketone camphor), and (+)-(3S)-linalyl pyrophosphate was the preferred substrate. With the (+)-bornyl pyrophosphate cyclase from S. officinalis, geranyl, neryl, and (-)-(3R)-linalyl pyrophosphates gave the expected (+)-(1R,4R)-stereoisomer as the sole product, and (-)-(3R)-linalyl pyrophosphate was the preferred substrate. However, (3S)-linalyl pyrophosphate yielded (-)-(1S,4S)-bornyl pyrophosphate, albeit at lower rates, indicating the ability of this enzyme to catalyze the anomalous enantiomeric cyclization.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-pinene and (+)- and (-)-camphene

Cyclase I from Salvia officinalis leaf catalyzes the conversion of geranyl pyrophosphate to the stereo-chemically related bicyclic monoterpenes (+)-alpha-pinene and (+)-camphene and to lesser quantities of monocyclic and acyclic olefins, whereas cyclase II from this plant tissue converts the same acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene and (-)-camphene as well as to lesser amounts of monocyclics and acyclics. These antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H]Linalyl pyrophosphate (3H:14C = 5.14) was tested as a substrate with both cyclases to determine the configuration of the cyclizing intermediate. This substrate with cyclase I yielded alpha-pinene and camphene with 3H:14C ratios of 3.1 and 4.2, respectively, indicating preferential, but not exclusive, utilization of the (3R)-enantiomer. With cyclase II, the doubly labeled substrate gave bicyclic olefins with 3H:14C ratios of from 13 to 20, indicating preferential, but not exclusive, utilization of the (3S)-enantiomer in this case. (3R)- and (3S)-[1Z-3H]linalyl pyrophosphate were separately compared to the achiral precursors [1-3H]geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. With cyclase I, geranyl, neryl, and (3R)-linalyl pyrophosphate gave rise exclusively to (+)-alpha-pinene and (+)-camphene, whereas (3S)-linayl pyrophosphate produced, at relatively low rates, the (-)-isomers. With cyclase II, geranyl, neryl, and (3S)-linalyl pyrophosphate yielded exclusively the (-)-isomer series, whereas (3R)-linalyl pyrophosphate afforded the (+)-isomers at low rates. These results are entirely consistent with the predicted stereochemistries and additionally revealed the unusual ability of these enzymes to catalyze antipodal cyclizations when presented with the unnatural linalyl enantiomer.     

Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.     

Biosynthesis of monoterpenes: stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to camphane and isocamphane monoterpenes

The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)-bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3S)-[1Z-3H]-linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:14C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The finding that the 1Z-3H of the linalyl precursor was positioned at the endo-alpha-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo- conformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids.     

Inhibition of Family II Pyrophosphatases by Analogs of Pyrophosphate and Phosphate

Imidodiphosphate (the pyrophosphate analog containing a nitrogen atom in the bridge position instead of oxygen) is a potent inhibitor of family II pyrophosphatases from Streptococcus mutans and Streptococcus gordonii (inhibition constant Ki approximately 10 microM), which is slowly hydrolyzed by these enzymes with a catalytic constant of approximately 1 min(-1). Diphosphonates with different substituents at the bridge carbon atom are much less effective (Ki = 1-6 mM). The value of Ki for sulfate (a phosphate analog) is only 12 mM. The inhibitory effect of the pyrophosphate analogs exhibits only a weak dependence on the nature of the metal ion (Mn, Mg, or Co) bound in the active site.     

Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita).

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.     

Evidence for the ionization steps in monoterpene cyclization reactions using 2-fluorogeranyl and 2-fluorolinalyl pyrophosphates as substrates

Conversion of geranyl pyrophosphate to cyclic monoterpenes is considered to involve the preliminary isomerization of this acyclic precursor to enzyme-bound linalyl pyrophosphate and the cyclization of this tertiary intermediate. 2-Fluorogeranyl pyrophosphate and 2-fluorolinalyl pyrophosphate are effective competitive inhibitors of the cyclization of geranyl pyrophosphate by several different monoterpene cyclases, and the electron withdrawing alpha-fluorine substituent was shown to suppress the rate of cyclic product formation from both tritium-labeled analogs by at least two orders of cyclic These results indicate that both steps of the coupled isomerization-cyclization sequence are initiated by ionization of an allylic pyrophosphate, and they confirm the electrophilic nature of this enzymatic reaction type and its similarity to the prenyltransferase reaction.     

Demonstration of a cyclic pyrophosphate intermediate in the enzymatic conversion of neryl pyrophosphate to borneol

A soluble enzyme preparation obtained from young sage (Salvia officinalis) leaves catalyzes the conversion of neryl pyrophosphate to (+)-borneol and the oxidation of (+)-borneol to (+)-camphor. Attempts to purify the borneol synthetase activity by gel permeation column chromatography resulted in the apparent loss of catalytic capability; however, subsequent recombination of column fractions demonstrated that two separable enzymatic activities were required for the conversion of neryl pyrophosphate to borneol. Several lines of evidence indicated that a water-soluble, dialyzable intermediate was involved in this transformation. The intermediate was isolated and subsequently identified as bornyl pyrophosphate by direct chromatographic analysis and by the preparation of derivatives and chromatographic analysis of both the hydrogenolysis (LiAlH₄) and enzymatic hydrolysis products of bornyl pyrophosphate. The results presented indicate that borneol is derived by cyclization of neryl pyrophosphate to bornyl pyrophosphate, followed by hydrolysis. This is the first demonstration of a cyclic pyrophosphorylated intermediate in the biosynthesis of bicyclic monoterpenes.     

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.     

Monoterpene cyclases. Stereoelectronic requirements for substrate binding and ionization

Enzymes from Salvia officinalis, capable of catalyzing the electrophilic isomerization and subsequent cyclization of geranyl pyrophosphate (3,8-dimethylocta-2E,6-dienyl pyrophosphate) to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate, were examined with a series of substrate analogs modified in carbon chain length and in the geometric and electronic character of the C2-C3 and C6-C7 olefinic domains. Inhibition studies with these monoterpene cyclases indicated that the pyrophosphate ester function was the principal determinant of substrate recognition and that the C2-C3 olefin was recognized largely on the basis of geometry, whereas the primary basis of interaction with the C6-C7 olefin was electronic. A related group of allylic pyrophosphates was tested for the ability to undergo enzyme-catalyzed ionization to afford olefinic and/or alcoholic products. From the relative reaction rates it was deduced that the alignment of the allylic pi-system with the C1-OP bond was essential for ionization of the substrate and that specific interaction with the distal C6-C7 isopropylidene function served not only to optimize orbital alignment but also to exclude water from the active site, and thus determine the partitioning of cationic intermediates into olefins or alcohols. From the combination of results, the interrelationships of substrate functional groups within the active site could be approximated and the topology of geranyl pyrophosphate binding to the cyclase thereby formulated.     

Mechanism of the pyrophosphate migration in the enzymatic cyclization of geranyl and linalyl pyrophosphates to (+)- and (-)-bornyl pyrophosphates

Soluble enzymes from sage (Salvia officinalis) and tansy (Tanacetum vulgare), which catalyze the cyclization of geranyl pyrophosphate and the presumptive intermediate linalyl pyrophosphate to the (+) and (-) enantiomers, respectively, of 2-bornyl pyrophosphate, were employed to evaluate mechanistic alternatives for the pyrophosphate migration in monoterpene cyclization reactions. Separate incubation of [1-3H2,alpha-32P]- and [1-3H2,beta- 32P]geranyl and (+/-)-linalyl pyrophosphates with partially purified preparations of each enantiomer-generating cyclase gave [3H, 32P]bornyl pyrophosphates, which were selectively hydrolyzed to the corresponding bornyl phosphates. Measurement of 3H:32P ratios of these monophosphate esters established that two ends of the pyrophosphate moiety retained their identifies in the cyclization of both precursors to both products and also indicated that there was no appreciable exchange with exogenous inorganic pyrophosphate in the reaction. Subsequent incubations of each cyclase with [8,9-14C,1-18O]geranyl pyrophosphate and with (1E)-(+/-)-[1-3H,3-18O]linalyl pyrophosphate gave the appropriate (+)- or (-)-bornyl pyrophosphates, which were hydrolyzed in situ to the corresponding borneols. Analysis of the derived benzoates by mass spectrometry demonstrated each of the product borneols to possess an 18O enrichment essentially identical with that of the respective acyclic precursor. The absence of P alpha-P beta interchange and the complete lack of positional 18O isotope exchange of the pyrophosphate moiety are compatible with tight ion pairing of intermediates in the coupled isomerization-cyclization of geranyl pyrophosphate and establish a remarkably tight restriction on the motion of the transiently generated pyrophosphate anion with respect to its cationic terpenyl reaction partner.     

Monoterpene cyclases: physicochemical features required for pyrophosphate binding determined from inhibition by structural analogs

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate, the ubiquitous C10 intermediate of isoprenoid biosynthesis, to a variety of monoterpene skeletons, and the pyrophosphoryl moiety is a primary determinant for substrate binding by these enzymes. To determine what specific features of this functional group are critical for enzymatic recognition, inorganic pyrophosphate and a series of structurally related analogs were examined as inhibitors of geranyl pyrophosphate:(+)-alpha-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from sage (Salvia officinalis). Analysis of trends in the magnitude of inhibition by the analogs relative to inorganic pyrophosphate indicated that the combination of ionization state (formal charge) at the enzymatic pH optimum, ability to chelate divalent metal ions, and intramolecular flexibility is required for effective interaction with both cyclases. Only when all of these criteria are met is inhibition of cyclization comparable to that observed with inorganic pyrophosphate.     

Conversion of [1-3H2,G-14C]geranyl pyrophosphate to cyclic monoterpenes without loss of tritium

Soluble enzyme preparations from Salvia officinalis convert the acyclic precursor [1-³H₂,G-¹⁴C]geranyl pyrophosphate to cyclic monoterpenes of the pinane (α-pinene,β-pinene), isocamphane (camphene), p-menthane (limonene,1,8-cineole), and bornane (bornyl pyrophosphate, determined as borneol) type without loss of tritium, and without significant conversion to other free acyclic intermediates. Similarly, [1-³H₂,G-¹⁴C]geraniol is converted in intact S. officinalis leaves to the cyclic monoterpene olefins and 1,8-cineole, as well as to isothujone and camphor, without loss of tritium from C(1). These results clearly eliminate transcis isomerization of geranyl pyrophosphate to neryl pyrophosphate via aldehyde intermediates prior to cyclization, and they support a scheme whereby the trans precursor is cyclized directly by way of a bound linaloyl intermediate.     

Gossypol inhibition of adenylate cyclase

Gossypol, a polyphenolic binaphthalene -dialdehyde reputed to exert contraceptive action in males, reversibly inhibits adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] in a concentration-dependent manner. In membranes prepared from a variety of organs, the half-maximal inhibitory concentration (IC50) ranges from 75 microM (rat Leydig tumor cells) to 250 microM (rat liver membranes). Kinetic studies using partially purified catalytic subunit isolated from bovine testis show that gossypol is competitive with ATP with an apparent Ki of 110 microM. These data suggest that gossypol inhibition of adenylate cyclase is due to direct interaction at the nucleotide-binding domain of the catalytic subunit of the enzyme.     

New inhibitors of sepiapterin reductase. Lack of an effect of intracellular tetrahydrobiopterin depletion upon in vitro proliferation of two human cell lines.

N-Acetylserotonin (compound 1) and N-acetyldopamine (compound 7) inhibit bovine adrenal medullary sepiapterin reductase in a manner competitive with the pterin substrate and have Ki values of 0.12 and 0.4 microM, respectively. Molecular modeling suggests that the phenyl rings of the two compounds bind in the pyrimidine pocket of the enzyme with the 3-hydroxyl of dopamine or the 5-hydroxyl of serotonin aligned at the pyrimidine 4-position. Further, the acetyl moieties of the two inhibitors appear to mimic the substrate side chain. Consistent with this analysis, N-acetyl-m-tyramine (compound 13) is also an excellent competitive inhibitor (Ki = 0.13 microM), whereas N-acetyltryptamine (compound 2), N-acetyl-p-tyramine (compound 14) and N-acetylphenylethylamine (compound 15) all bind poorly. Interestingly, restricted-rotation analogs of N-acetyldopamine and N-acetyl-m-tyramine are noncompetitive inhibitors of the enzyme. Modification of N-acetyldopamine to N-chloroacetyldopamine (compound 10) or of N-acetylserotonin to the N-chloroacetyl (5) or N-methoxyacetyl (compound 6) analogs results in greatly increased competitive affinity, with Ki = 0.014 microM for the dopamine analog and 0.006 and 0.008 microM, respectively, for the serotonin analogs. In MOLT-4 T-cell leukemia and MCF-7 breast adenocarcinoma in culture, 0.1 mM N-methoxyacetylserotonin depleted tetrahydrobiopterin by greater than or equal to 97 and greater than 50%, respectively, with no effect upon cell growth. In both cell lines, the GTP cyclohydrolase inhibitor, 2,4-diamino-6-hydroxypyrimidine at 1-5 mM also depleted tetrahydrobiopterin greater than or equal to 97%. In this case, however, modest growth inhibition did occur. Since the growth inhibition could not be reversed upon tetrahydrobiopterin repletion, inhibition was due to other effects of the inhibitor rather than to tetrahydrobiopterin depletion. The results show that there is no effect on cell growth when at least 97% of the tetrahydrobiopterin in these cell lines is depleted. Since the sepiapterin reductase inhibitor depleted tetrahydrobiopterin with fewer nonspecific effects than the cyclohydrolase inhibitor, it will be useful for determining metabolic effects of tetrahydrobiopterin depletion.