Liliana Chayet

Biosynthesis of monoterpene hydrocarbons from [1-3H]neryl pyrophosphate and [1-3H]geranyl pyrophosphate by soluble enzymes from Citrus limonum

Abstract A soluble enzyme preparation from the flavedo of Citrus limonum transforms [1- 3 H 1 ]neryl pyrophosphate or [1- 3 H 1 ]geranyl pyrophosphate into β-pinene, sabinene, α-pinene, and limonene. The enzyme has been partially purified and stabilized by precipitation with polyethyleneglycol. The enzymic cyclization requires the presence of Mn 2+ , which cannot be replaced with Mg 2+ . The addition of reagents containing sulfhydryl groups is essential for optimal activity. Allylic C 10 monophosphates do not act as substrates, but they inhibit hydrocarbon formation. Inorganic pyrophosphate has a similar inhibitory effect. No interconversion of neryl and geranyl pyrophosphate has been observed. Possible pathways for the enzymic cyclization reactions are proposed.

Biosynthesis of sesquiterpene alcohols and aldehydes by cell free extracts from orange flavedo

Abstract Water soluble enzymes obtained from orange flavedo form two isomeric farnesols from 1- 3 H labeled geranylpyrophosphate plus isopentenylpyrophosphate

Human placental atp-diphosphohydrolase: Biochemical characterization, regulation and function

1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M(r) and pI values of both ATPase-ADPase activities. 2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher. 3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DES and slightly by DCCD. 4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A. 5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of -SH, COO-, -OH, and probably also Tyr and Trp. 6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes. 7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

Enhancement of the hydrolysis of geranyl pyrophosphate by bivalent metal ions. A model for enzymic biosynthesis of cyclic monoterpenes

Hydrolysis of geranyl pyrophosphate is catalyzed by salts of Mn2+ and involves C-O bond cleavage. The first order rate constants reach limiting values with [Mn2+] > 10−2 M, and the most reactive species is GPP(Mn2+)2 at the optimum pH of 6.5–7. The products are similar to those from acid hydrolysis except that more cyclic hydrocarbons are formed in the presence of metal ions. Hydrolysis of geranyl phosphate is inhibited, and that of citronnellyl pyrophosphate is weakly catalyzed by Mn2+. Other divalent metal cations catalyze the hydrolysis of geranyl pyrophosphate and the sequence of effectiveness is Cu2+ > Mn2+ > Zn2+ > Co2+ < Mg2+ ~ Ca2+.

Complexes of bivalent cations with neryl and geranyl pyrophosphate: Their role in terpene biosynthesis

Abstract Kinetic analysis of the nonenzymic solvolysis of neryl and geranyl pyrophosphate (NPP and GPP, respectively) showed that the dissociation constants of the bis-metallic complexes with Mg 2+ and Mn 2+ were larger for NPP than for GPP by approximately one order of magnitude. Rate constants for reaction of the bis-metallic complexes were larger for NPP than for GPP. Qualitatively similar behavior was observed with complexes of Co 2+ . Extents of elimination and cyclization were increased by metal ions. Carbocyclase-catalyzed formation of cyclic monoterpene hydrocarbons in the presence of Mg 2+ involved bis-metallic complexes as the “true” substrates.

Prenyltransferases from the flavedo of Citrus sinensis

Abstract A prenyltransferase activity (EC 2.5.1.1) has been partially purified from the flavedo of Citrus sinensis with 30–40-fold purification and 35–60 % yield. The enzyme catalyses the condensation of IPP with DMAPP or GPP. The products are neryl and geranyl pyrophosphate as well as (2 E ,6 E )- and (2 Z ,6 E )-farnesyl pyrophosphate. The two C 15 -products are predominant. The E - and Z -synthetase activities are partially dissociated during the purification procedure, as well as by heat or ageing. Preparations devoid of Z -synthetase were obtained. Mg 2 + is required for full activity. Mn 2 + or Co 2 + can replace Mg 2 + . The ratio of E/Z -products formed is different for each cation. Mg 2 + complexes of allylic substrates or of products protect the enzyme against heat-inactivation and against inactivation by DTNB. The results are interpreted in terms of two or more prenyltransferases stereoselective for the synthesis of E - and Z -products.

Synthesis of isomeric farnesols by soluble enzymes from Pinus radiata seedlings

Abstract Water soluble enzymes obtained from Pinus radiata seedlings form two sesquiterpene alcohols from 2- 14 C mevalonic acid. They have been identified as 2,6- trans,trans -farnesol and 2- cis ,6- trans -farnesol. The pyrophosphate of the former prenol could be isolated, but there was no evidence of the presence of phosphorylated derivatives of the cis isomer. The same pair of sesquiterpene alcohols and trans -farnesyl pyrophosphate are formed from isopentenyl pyrophosphate plus geranyl pyrophosphate (2- trans ). Neryl pyrophosphate (2- cis ) is completely inactive as a precursor of farnesols. Isomerization of trans - to cis -farnesyl pyrophosphate did not occur in this system.

Synthesis of monoterpene hydrocarbons from [1-3H]linalyl pyrophosphate by carbocyclase from Citrus limonum☆

Abstract A partially purified enzyme preparation from the flavedo of Citrus limonum utilized [1- 3 H]linalyl pyrophosphate as a substrate for cyclic terpene hydrocarbon formation more efficiently than the pyrophosphates of nerol and geraniol. The products formed from all three substrates are α-pinene, β-pinene, limonene, and γ-terpinene. Neryl and geranyl pyrophosphate inhibit the formation of these products from linalyl pyrophosphate. No free linalyl pyrophosphate could be detected during the enzymatic formation of cyclic terpene hydrocarbons from geranyl pyrophosphate. Mn 2+ catalyzes the nonenzymatic solvolysis of linalyl pyrophosphate, forming myrcene and ocymenes and no bicyclic hydrocarbons. Linalyl pyrophosphate is a sterically plausible precursor of cyclic hydrocarbons, but the present data support only its role as an alternative substrate and not as an obligatory free intermediate in terpene biosynthesis.

Substrate and metal specificity in the enzymic synthesis of cyclic monoterpenes from geranyl and neryl pyrophosphate

A partially purified enzyme (carbocyclase) from the flavedo of Citrus limonum formed alpha-pinene, beta-pinene, limonene, and gamma-terpinene from geranyl pyrophosphate (GPP) and neryl pyrophosphate. The maximum specific activities obtained were 7.0 and 3.6 nmol/min/mg, respectively. Cross-inhibition by the two substrates were observed and the ability to utilize neryl pyrophosphate was almost completely lost with aging. Citronellyl pyrophosphate and dimethylallyl pyrophosphate were the most effective inhibitors of carbocyclase. Isopentenyl pyrophosphate, the monophosphate esters of nerol and geraniol, as well as inorganic pyrophosphate were much less effective inhibitors. The enzyme had an absolute requirement for Mn2+. It could be replaced with about 2% effectiveness by Mg2+ and Co2+. Kinetic studies showed that the observed reaction rate correlates with the calculated concentration of the GPP (Mn2+)2 species. Previous evidence with nonenzymatic reactions and the results presented support the view that the mechanism of carbocyclase may be the intramolecular analog of prenyltransferase.

ATP-DIPHOSPHOPHYDROLASE ACTIVITY IN RAT HEART TISSUE

Extracellular nucleotides interact with specific receptors on the cell surface and are locally metabolized by ecto‐nucleotidases. Biochemical characterization of the ATPase and ADPase activities detected in rat heart sarcolemma, under conditions where mitochondrial ATPase and adenylate kinase were blocked, supports our proposal that both activities correspond to a single enzyme, known as ATP‐diphosphohydrolase or apyrase. The physiological function of this enzyme could be dephosphorylation of the nucleotides present in the interstitial heart compartment acting together with 5′‐nucleotidase. Both hydrolytic activities have similarities in: sarcolemma localization, bivalent metal ion dependence, optimum pH, effect of several amino acid residue modifiers, competitive inhibition of nucleotide analogs, and broad nucleoside di‐and triphosphate specificity. The ATPase activity could not be separated from the ADPase either through isoelectrofocusing or electrophoresis under acid conditions.