SPECTROSCOPIC AND KINETICS STUDIES OF A HIGH-SALT-STABILIZED FORM OF THE PURPLE ACID-PHOSPHATASE FROM BOVINE SPLEEN
SPECTROSCOPIC AND KINETICS STUDIES OF A HIGH-SALT-STABILIZED FORM OF THE PURPLE ACID-PHOSPHATASE FROM BOVINE SPLEEN
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DOI：
10.1021/bi00226a007
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发表时间：
1991-03-26
期刊：
影响因子：
2.9
通讯作者：
AVERILL, BA
中科院分区：
文献类型：
--
作者：
VINCENT, JB;CROWDER, MW;AVERILL, BA
使用一种改进的纯化程序在高盐环境中保持该酶，从而分离出一种新形式的牛脾紫色酸性磷酸酶。这种酶不能通过电泳、等电聚焦、Western印迹分析或N-末端氨基酸序列与前面描述的[Davis，J.C.，Lin，S.S.，&Averill，B.A.(1981)BioChemical 20,4062]区分开来，并以还原的(粉红色)形式表现出相同的催化性质和EPR谱。然而，CD光谱表明，它确实具有更高的有序性，在氧化反应中表现出明显不同的反应活性，在抑制阴离子结合或改变pH时表现出不同的可见光谱。这种新的高盐稳定形式的酶的性质使人们能够广泛地检查氧化和还原的酶与抑制性阴离子的络合物的可见吸收光谱。根据它们对可见吸收最大值的影响及其对pH的敏感性，这些阴离子可分为三类：磷酸盐、砷酸盐和AMP；钨酸盐和钼酸盐；以及氟化物。通过对这些抑制剂存在下酶的稳态动力学的详细研究，加强了这种分组，发现第一类由于竞争和非竞争结合位点的存在而表现出混合型抑制，而第二类表现出简单的非竞争性抑制。氟化物表现出复杂的缓蚀行为，其特征是Lineweaver-Burk曲线；这种行为不能归因于抑制性氟化铝络合物的存在。综上所述，光谱和动力学数据与四面体氧阴离子通过桥联双核中心的两个铁原子以非竞争方式结合的图像是一致的，较小的阴离子也能够以竞争方式结合在单个铁原子上。
Use of a revised purification procedure that maintains the enzyme in a high-salt environment has resulted in the isolation of a new form of the bovine spleen purple acid phosphatase. This enzyme cannot be distinguished from that previously described [Davis, J. C., Lin, S. S., & Averill, B. A. (1981) Biochemistry 20, 4062] by electrophoresis, isoelectric focusing, Western blot analysis, or N-terminal amino acid sequence and exhibits identical catalytic properties and EPR spectra in the reduced (pink) form. It does, however, possess a much more highly ordered structure as shown by CD spectra and exhibits markedly different reactivity upon oxidation and different visible spectra upon binding of inhibitory anions or changing pH. The properties of the new high-salt-stabilized form of the enzyme have permitted an extensive examination of the visible absorption spectra of complexes of the oxidized and reduced enzyme with inhibitory anions. It is found that these anions may be grouped into three classes on the basis of their effect on the visible absorption maximum and their sensitivity to pH: phosphate, arsenate, and AMP; tungstate and molybdate; and fluoride. This grouping is reinforced by a detailed examination of the steady-state kinetics of the enzyme in the presence of these inhibitors, which reveals that the first class exhibits mixed-type inhibition due to the presence of competitive and noncompetitive binding sites, while the second class exhibits simple non-competitive inhibition. Fluoride exhibits complex inhibition behavior characterized by curved Lineweaver-Burk plots; this behavior cannot be attributed to the presence of inhibitory aluminum fluoride complexes. Taken together, the spectral and kinetics data are consistent with a picture in which tetrahedral oxyanions bind in a noncompetitive fashion by bridging the two iron atoms in the dinuclear center, with the smaller anions also being able to bind in a competitive manner at a single iron atom.