氨基酸对鲍鱼碱性磷酸酶活力的影响

选用L Ala、L Val、L Leu等15种氨基酸为效应物,研究它们对杂色鲍(Haliotisdiversicolor)碱性磷酸酶(EC.3.1.3.1)催化pNPP水解反应的影响.结果表明:上述这些氨基酸对鲍鱼碱性磷酸酶均有一定程度的抑制作用,抑制程度随着试剂浓度增大而加剧.当浓度为10.0mmol/L时,分别可以使酶活力下降:7.77%、36.75%、22.79%、39.86%、16.16%、19.33%、94.77%、20.19%、17.02%、40.15%、31.08%、17.52%、30.56%、36.77%和12.59%.尤其以L Cys对酶的抑制作用最为显著(抑制率为94.77%),其次是L Ile和L His,它们均能使酶活力下降40%;对酶抑制率在30%以上的氨基酸还有L Val、L Trp、L Phe和L Lys,其它选用的氨基酸对酶的抑制率小于25%.选择对杂色鲍ALP具有较明显的抑制作用的氨基酸L Val、L Ile、L Trp和L Cys等氨基酸为研究对象,研究它们对酶催化pNPP水解反应的抑制作用机理,并测定其抑制常数.结果表明L Cys的抑制作用为混合型效应,其KI和KIS值分别为0.042mmol/L和0.139mmol/L;而L Val、L Ile、L Trp为反竞争性,其KIS分别为9.56、8.55、8.09mmol/L.

Inhibition of Alkaline Phosphatase by Amino Acids

Alkaline phosphatase (ALP,EC3.1.3.1)is a metalloenzyme which catalyzes the nonspecific hydrolysis of phosphate monoesters.It is also concerned with the absorption of phosphate and calcium from seawater and exuviating cell of Haliotis diversicolor.The effects of some amino acids on the activity of alkaline phosphatase from Haliotis diversicolor were studied.The results showed that all the amino acids tested had inhibitory effects on the enzyme activity, and that the inhibitory intensity enhanced with increasing the concentrations of amino acids.At the concentration reached to 10.0 mmol/L,the inhibition ratios were determined to be 94.77%(L-Cys),40.15%(L-His),39.86%(L-Ile),36.77%(L-Trp),36.75%(L-Val),31.08%(L-Lys),30.56%(L-Phe),22.79%(L-Leu),20.19%(L-Glu),19.33%(L-Thr),17.52%(L-Arg),17.02%(L-Asp),16.16%(L-Ser),12.59%(L-Pro)and 7.77%(L-Ala).The inhibitory kinetics of L-Cys,L-Val,L-Ile,L-Trp,on the enzyme were further studied.The results showed that the inhibition of L-Cys on the enzyme is found to be a mixed type.The inhibition constants of free enzyme(K_I)and enzyme-substrate complex(K_(IS))by L-Cys were determined to be 0.042 mmol/L and 0.139 mmol/L,respectively.L-Val,L-Ile,and L-Trp were found to be uncompetitive inhibitors of the enzyme,and their inhibition constants(K_(IS))were determined to be 9.56,8.55 and 8.09 mmol/L,respectively.