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Synthesis of Aspartame by Thermolysin: An X-ray Structural Study.

ABSTRACT: Protease mediated peptide synthesis (PMPS) was first described in the 1930s but remains underexploited today. In most PMPS, the reaction equilibrium is shifted toward synthesis by the aqueous insolubility of product generated. Substrates and proteases are selected by trial and error, yields are modest, and reaction times are slow. Once implemented, however, PMPS reactions can be simple, environmentally benign, and readily scalable to a commercial level. We examined the PMPS of a precursor of the artificial sweetener aspartame, a multiton peptide synthesis catalyzed by the enzyme thermolysin. X-ray structures of thermolysin in complex with aspartame substrates separately, and after PMPS in a crystal, rationalize the reaction's substrate preferences and reveal an unexpected form of substrate inhibition that explains its sluggishness. Structure guided optimization of this and other PMPS reactions could expand the economic viability of commercial peptides beyond current high-potency, low-volume therapeutics, with substantial green chemistry advantages.

SUBMITTER: Birrane G

PROVIDER: S-EPMC4060929 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Synthesis of Aspartame by Thermolysin: An X-ray Structural Study.

Birrane Gabriel G Bhyravbhatla Balaji B Navia Manuel A MA

ACS medicinal chemistry letters 20140410 6

Protease mediated peptide synthesis (PMPS) was first described in the 1930s but remains underexploited today. In most PMPS, the reaction equilibrium is shifted toward synthesis by the aqueous insolubility of product generated. Substrates and proteases are selected by trial and error, yields are modest, and reaction times are slow. Once implemented, however, PMPS reactions can be simple, environmentally benign, and readily scalable to a commercial level. We examined the PMPS of a precursor of the ...[more]

PMID: 24944748

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Project description:BackgroundAspartame is a commonly used intense artificial sweetener, being approximately 200 times sweeter than sucrose. There have been concerns over aspartame since approval in the 1980s including a large anecdotal database reporting severe symptoms. The objective of this study was to compare the acute symptom effects of aspartame to a control preparation.MethodsThis was a double-blind randomized cross over study conducted in a clinical research unit in United Kingdom. Forty-eight individual who has self reported sensitivity to aspartame were compared to 48 age and gender matched aspartame non-sensitive individuals. They were given aspartame (100mg)-containing or control snack bars randomly at least 7 days apart. The main outcome measures were acute effects of aspartame measured using repeated ratings of 14 symptoms, biochemistry and metabonomics.ResultsAspartame sensitive and non-sensitive participants differed psychologically at baseline in handling feelings and perceived stress. Sensitive participants had higher triglycerides (2.05 ± 1.44 vs. 1.26 ± 0.84mmol/L; p value 0.008) and lower HDL-C (1.16 ± 0.34 vs. 1.35 ± 0.54 mmol/L; p value 0.04), reflected in 1H NMR serum analysis that showed differences in the baseline lipid content between the two groups. Urine metabonomic studies showed no significant differences. None of the rated symptoms differed between aspartame and control bars, or between sensitive and control participants. However, aspartame sensitive participants rated more symptoms particularly in the first test session, whether this was placebo or control. Aspartame and control bars affected GLP-1, GIP, tyrosine and phenylalanine levels equally in both aspartame sensitive and non-sensitive subjects.ConclusionUsing a comprehensive battery of psychological tests, biochemistry and state of the art metabonomics there was no evidence of any acute adverse responses to aspartame. This independent study gives reassurance to both regulatory bodies and the public that acute ingestion of aspartame does not have any detectable psychological or metabolic effects in humans.Trial registrationISRCTN Registry ISRCTN39650237.

Project description:Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.

Project description:BackgroundPhenylketonuria (PKU) is a rare genetic metabolic disorder in which especially high phenylalanine (Phe) concentrations cause brain dysfunction. If untreated, this brain dysfunction results in severe microcephaly, intellectual disability, and behavioral problems. Dietary restriction of Phe is the mainstay of PKU treatment, with long-term successful outcomes. Aspartame, an artificial sweetener sometimes added into medications, is metabolized in the gut into Phe. Then, patients suffering from PKU on a Phe-restricted diet should avoid consumption of aspartame. The aim of our study was to evaluate the number of drugs containing aspartame and/or Phe as an excipient, and to quantify their corresponding Phe intake.MethodsThe list of drugs marketed in France containing aspartame and/or Phe was established using a national medication database called "Theriaque". For each drug, the corresponding daily Phe intake was calculated according to age and weight and was distributed into 3 categories: high (> 40 mg/d), medium (10 to 40 mg/d) and low (< 10 mg/d) Phe intake.ResultsThe number of drugs containing Phe or its precursor aspartame remained very limited (n = 401). Among the aspartame containing drugs, Phe intakes were significant (medium or high) for only half of them whereas there were negligible for the others. Furthermore, these medications with a significant Phe intake were limited to few pharmaceutical classes (mainly antiinfectives agents, analgesics, and drugs for nervous system), and within these classes the drugs were limited to a small number of molecules, including principally amoxicillin, amoxicillin + clavulanic acid and paracetamol/ acetaminophen.DiscussionIn situations requiring the use of these molecules, we propose as an alternative, the use of an aspartame-free form of these molecules or a form with a low Phe intake. If it is not possible, we propose as second-line the use of another antibiotics or analgesics. Finally, we have to remember the benefits-risk balance to use medications containing significant Phe intake in PKU patients. Indeed, it may be better to use a Phe containing medication in the absence of an aspartame-free form of this drug rather than to leave a person with PKU without treatment.

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Synthesis of Aspartame by Thermolysin: An X-ray Structural Study.

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Synthesis of Aspartame by Thermolysin: An X-ray Structural Study.

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