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KMID : 0613820090190091226
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Journal of Life Science
2009 Volume.19 No. 9 p.1226 ~ p.1231
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Biochemical Characterizations of Phenylalanine Ammonia-Lyase and its Mutants to Develop an Enzymatic Therapy for Phenylketonuria
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Kim Woo-Mi
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Abstract
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Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the bi℃hemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its Vmax values for phenylalanine and tyrosine were 1.77 and 0.47 μ㏖/ min/㎎ protein, respectively, and its Km values were 4.77×10?⁴ and 4.37×10?⁴ M, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at -80℃ for 5 months and possessed 93.4% of its activity under 4℃ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild- type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.
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KEYWORD
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Phenylketonuria, phenylalanine ammonia-lyase, chymotrypsin, trypsin, proteolysis
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