Tipos de “misa” y simbología

The haem biosynthesis pathw ay requires, in animals, the action of eight enzym es to produce haem from succinyl CoA and glycine. The

reduction in activity of any of these enzym es will lead to a deficiency in haem production and ultimately result in disease. The decrease in activity in the

first of these enzymes, the rate controlling 5-aminolaevulinic acid synthase, causes the disorder of hereditary sideroplastic anaemia (Bottomley, 1982). The decrease in activity in any of the rem aining seven enzym es of the pathw ay leads to a group of metabolic disorders know n as porphyrias (see Elder and

Path, 1982, for an overview). Porphyrias are brought about by the

accumulation of the haem precursors, resulting in characteristic clinical and biochemical features (Bottomley and Muller-Eberhard, 1988). There are seven docum ented hum an porphyrias, each representing a defect in one of the enzymes.

The third enzyme in the haem biosynthesis pathw ay is

porphobilinogen deaminase, and the failure of this enzym e to turnover

substrate has a two-fold effect upon physiological conditions. Firstly, the pool of tetrapyrroles available for haem synthesis will diminish, limiting the

availability of haem for its various functions as a prosthetic group. In

hum ans, the decrease in haem levels will also lessen the regulatory effect that haem has on controlling 5-aminolaevulinic acid synthase, leading to increased concentrations and activity of the enzyme (Kappas, 1983). Secondly, the

newly synthesised 5-aminolaevulinic acid synthase and increased activity combine to lead to concentrations of porphobilinogen w hich are in excess of the m alfunctioning enzyme's ability to turn over, resulting in the

accumulation of the haem precursors 5-aminolaevulinic acid and

porphobilinogen. The hum an diseased state arising from the deficiency of porphobilinogen deaminase activity is term ed acute interm ittent porphyria (AIP).

The haem deficiency is thought to m anifest itself indirectly. The haem requiring enzyme hepatic tryptophan pyrrolase converts tryptophan to

Chapter 1 : Introduction

tryptophan will rise, stimulating the synthesis of the neurotransm itter 5- hydroxytryoptamine. The hypothesis is supported by the increased excretion of indoles by AIP patients in their urine (Kappas, 1983). Neurological

disorders are also believed to be brought about by the increased concentrations of the haem precursors 5-aminolaevulinic acid and

porphobilinogen, and both 5-aminolaevulinic acid and porphobilinogen have been identified in the cerebrospinal fluid of patients w ith AIP (Sweeney et ah, 1970). In support, there are many reports of the effects of in vitro

adm inistration of the two precursors, although the concentrations used in the majority of cases have not been physiologically significant. A final theory of the possible effect of malfunctioning porphobilinogen deam inase is one where the overactivity of 5-aminolaevulinic acid synthase leads to a depletion in its coenzyme, pyridoxal phosphate. Again, plasma pyridoxal

concentrations have been found to be lower in AIP patients, but no

correlation has been found between coenzyme concentrations and clinical activity of the disease (Hamfelt and W etterberg, 1969). It should be noted that all of the above hypotheses are contested, and therefore m ust rem ain only theories.

AIP, like most porhyrias, is inherited in an autosomal dom inant m anner (Kappas, 1989). In affected individuals, the m ean activity of the enzyme is 50% of the norm al (Strand et ah, 1972), which is in keeping w ith the heterozygous state of these subjects. It is the most common of the porphyrias w ith an estim ated frequency of 1 per 10,000 persons. The frequency of the disorder rises to 1 per 500 amongst psychiatric patients, which is predictable considering the neurological symptons brought on by the m alfunctioning enzyme. The disease manifests typically in acute attacks, and in addition to the neurological disorders of psychiatric involvement, peripheral neuritis and paralysis, the sym ptoms include acute abdominal pain, vom itting and

constipation. The elevated am ounts of haem precursors result in a diagnostic 'port w ine red' colouring of the urine on air oxidation, indicative of the presence of porphobilin, an auto-oxidised product of porphobilinogen.

Clinically, haem arginate is administered therapeutically during acute attacks, as exogenous haem will relieve the feedback activation of 5-aminolaevulinic acid synthase. Fortunately, only 10% of know n AIP heterozygotes are

sym ptomatic, and the disease is precipitated by factors such as poor diet, alcohol, certain drugs and hormones.

1.8.2 H u m a n A IP M u ta tio n s a n d th e Structure o f P o r p h o b ilin o g e n D e a m in a se

The increasing use of polymerase chain reaction (PCR ) m ethods over recent years has allowed the genetic lesions responsible for AIP to be

identified. PCR has been employed to amplify m utant DNA for sequence analysis and hence detection of m utations (Picat et al., 1991; M gone et al., 1992; Delfau et al., 1990; Llewellyn et al., 1992; Scobie et al., 1990). The m utant

proteins can be classed according to their enzymic activity and their

immunological cross-reactivity, allowing a useful phenotypic characterisation of AIP carriers to be m ade (Desnick et at., 1985; N ordm ann et al.. 19901. A

positive GRIM status (cross im m uno reacting material) indicates a m u tan t allele w hich expresses recognisable protein but displays reduced activity, and a CRIM-negative status indicates an unrecognisable protein.

The determ ination of the three-dimensional structure of E. coli

porphobilinogen deam inase (Louie et ah, 1992) allows the m olecular basis of AIP to be better understood. There is 43% identity betw een the E. coli and hum an sequences of porphobilinogen deaminase (rising to 60% for

conserved residues), and the structure of the enzyme from E. coli can therefore act as a suitable model for the hum an enzyme. In this way, the reported hum an m utations have been analysed for their likely effects on the enzym e structure and catalytic process (Brownlie et ah, 1995). The E. coli m odel of the enzym e does have limitations to its use, both in mobile regions and also in "insert" regions where the structure of the hum an enzym e can only be predicted. However, analysis of intron/extron boundaries indicates that all introns are located in or near to loops connecting secondary structural elements and therefore, overall, the E.coli enzyme structure is unlikely to be significantly different to that of the hum an enzyme.

The m utations of porphobilinogen deaminase identified in AIP

sufferers can be classified into one of four types. The first of these, the type 1 m utations, are those which result in frameshifts, incorrect splicing and inappropriate stop codons. These factors easily explain the CRIM-negative status that all b u t one of the type 1 m utations display. The m utant proteins are predictably unable to fold in a recognisable m anner, and m ay additionally be m ore susceptible to rapid proteolysis.

Chapter 1 : Introduction

Type 2 m utations destabilise the protein structure through steric hindrance or loss of interactions. For example, two m utations of an invariant arginine, R116W and R116T [101], are obviously detrim ental to the protein and result in a CRIM-negative status (hum an num bering, followed by E.coli num bering in brackets). The arginine is located on one of two short strands linking dom ains 1 and 2, and the charged side-chain participates in an ion- pair w ith a glutamate, E250. The effect of disrupting the R116-E250 salt bridge is also confirmed by the CRIM-negative status of the hum an E250K m utant. Surprisingly, the site-directed m utations at this position in the E.coli enzyme [RIOIH or RIOIL] are not disruptive to the enzyme. Type two m utations may also involve m utations in the hydrophobic core, which are deleterious to the protein structure, either because they introduce a buried charge group or lead to large volume change and associated steric clashes, e.g. L177R [159] and A31T [16].

Type 3 m utations involve residues im portant to the catalytic reaction, and are easily reconciled w ith the disruption caused. The arginine residues R149, R150, R167 and R173 [131,132,149 and 155] all form im portant interactions w ith the acetate and propionate side groups of the cofactor and their m utations lead to reduced catalytic activity. A m utation of R149 to histidine or leucine (R149H or R149L) causes the loss of interaction w ith ring C l of the cofactor and gives rise to the apoenzyme form. The E.coli

apoenzym e is unstable, and the hum an apoenzyme is probably equally rapidly degraded, explaining the CRIM-negative status of R149 m utants. Not all m utants in the catalytic site lead to unrecognisable proteins. The m utation of residue R167, which interacts with the ring C2 side chain w hen it occupies the hypothesised substrate-binding site, to histidine or leucine (R167H or R167L) leads to an accumulation of the ES intermediate and an elevated Both m utants R167H and R167L have a CRIM-positive status, as do m utants of R173.

Finally, there are type 4 m utations which have an unknow n effect. Such m utations are neither exclusively CRIM-positive nor CRIM-negative. All type 4 m utations are found on the surface of the molecule.

In general, around 85% of unrelated patients w ith AIP have a CRIM- negative phenotype. The major changes in structure which bring about this phenotype can explain the loss of activity. Of the CRIM-positive mutations.

most occur around the large active site. Here, a num ber of m utations m ay occur w hich w ould be detrimental for activity b u t not affect the structure. The high CRIM -positive/activity ratios of some m utants m ay be satisfied by the presence of stable enzyme-intermediates, which become 'trapped' by the inability of the m utant enzyme to carry the reaction further.

1.9 C om parisons B etw een P orp h ob ilin ogen D eam in ase and