Catalytic Properties Of Human α-hydroxy Acid Dehydrogenase

L-lactate dehydrogenase（L-LDH）and L-malate dehydrogenase（L-MDH）are the two most common L-α-hydroxy acid dehydrogenases in human body,which can stereoselectively catalyze the reversible reaction betweenα-keto acid andα-hydroxy acid.α-hydroxy acids are a kind of important physiologically active substances,which play an important role in antibacterial,anti-inflammatory and antithrombotic activities.Among them,aromaticα-hydroxy acids,such as phenyllactic acid and its structural analogues,p-hydroxyphenylactic acid and 3,4-dihydroxyphenyllactic acid,have been proved to be related to many metabolic diseases in human body.Therefore,it is of great significance to explore the metabolic source ofα-hydroxy acids in human body.In this study,the reduction characteristics of human L-lactate dehydrogenase and L-malate dehydrogenase onα-keto acid were systematically determined,and L-lactate dehydrogenase and L-malate dehydrogenase with catalytic activity on aromaticα-keto acid were screened out,and their enzymatic properties were studied.I In addition,in this study,porcine heart was used as purified raw material,and it was confirmed that pig heartα-hydroxyate dehydrogenase can catalyze the production of L-aromaticα-hydroxyate,which further confirmed that the catalysis ofα-hydroxyate dehydrogenase is one of the metabolic sources of L-aromaticα-hydroxyate in human body.The main results are as follows:（1）In addition to natural substrates,human L-lactate dehydrogenase and L-malate dehydrogenase also have catalytic activities for a variety ofα-ketoacids,among which LDHC,LDHL6A and MDH1 can catalyze phenylpyruvic acid,p-hydroxyphenylpyruvic acid and3,4-dihydroxyphenylpyruvic acid to generate corresponding aromaticα-hydroxy acids.（2）The enzymatic properties of LDHC,LDHL6A and MDH1 were investigated.The optimum temperatures of the three enzymes were 37℃,35℃and 45℃,and the optimum p H was 6.5,6.5 and 5.5.The kinetic parameters of p-phenylpyruvic acid,p-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid in LDHC,LDHL6A and MDH1 were investigated.The K_mof LDHC p-phenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid were 0.90 m M and 0.92 m M;LDHL6A has high affinity for phenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid,with K_mof 0.77 m M and 0.80 m M;MDH1 has a very high affinity（K_m=0.46 m M）and catalytic efficiency(K_cat/K_m=23.87m M^-1·S^-1)for p-hydroxyphenylpyruvate,a high affinity for 3,4-dihydroxyphenylpyruvic acid,and a low affinity for phenylpyruvic acid（K_m=3.76 m M）.（3）Porcine heartα-hydroxy acid dehydrogenase was purified from pig heart,and it was proved to be a single band with a molecular weight of about 36 k Da by SDS-PAGE protein gel electrophoresis,which confirmed that porcine heartα-hydroxyacid dehydrogenase could catalyze the formation of L-aromaticα-hydroxyacid.