Abstract
Incubation of leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with various concentrations of Mg2+ at various pH values in 1 M KCl and 0.155 M trimethylamine-HCl at 37 degrees confirms that Mg2+ competes with Zn2+ for binding only 1 site per 54,000-dalton subunit. The ratio of the apparent association constants (1KZn:1KMg = 1KZn/Mg) at this site (site 1) was estimated to be 20,720 at pH 8.16, 10,570 at pH 8.44, 3,590 at pH 8.78, and 660 AT PH 9.14. The decrease in values of 1KZn/Mg with increasing pH in the activation of leucine aminopeptidase by Mg2+ is attributed to the lowering of the free Zn2+ concentration relative to that of free Mg2+ caused by the formation of ZnOH+ and Zn(OH)2 complexes with increasing OH- concentration. When corrections are made for the binding of Zn2+ by OH- ions, the pH-independent ratio of association constants (1KZn:1KMg = 1KZn/Mg) for the relative binding of Zn2+ and Mg2+ at site 1 of leucine aminopeptidase in 29,800. From the effect of pH on the relative binding constant, a value (beta2) for the product of the two stepwise association constants for the formation of Zn(OH)2 from Zn2+ and OH- (Zn2+ + OH- in equilibrium ZnOH+; ZnOH+ + OH- in equilibrium Zn(OH)2) was estimated to be 4.42 X 10(10) M-2 at 37 degrees. Values of Km at pH 7.5 AND 30 degrees with L-leucine p-nitroanilide as substrate in the presence of 0.01 M NaHCO3 are 4.13 and 2.01 mM for the zinc-zinc and magnesium-zinc enzymes, respectively. Values for Vmax are 0.2 and 2.49 mumol/min/mg, respectively.
Highlights
The decrease in values of ‘K.,n,Mg with increasing pH in the activation of leucine aminopeptidase by Mg *+ is attributed to the lowering of the free Zn*+ concentration relative to that of free Mg2+ caused by the formation of ZnOH+ and Zn(OH), complexes with increasing OH
When corrections are made for the binding of Zn*+ by OH- ions, the pH-independent ratio of association constants ( ‘Kzn:‘K& = ‘K&,) for the relative binding of Znz+ and Mgz+ at site 1 of leucine aminopeptidase is 29,800
A number of investigations have shown that Mn*+ is an activator [3], that the activation is time-dependent [4, 5], and that activation may be attributed to the binding of the metal ion to the protein [6, 7]. The latter conclusion, based on the observation that specific activity increased in the presence of increasing free Mgz+ or MnZ+ concentrations [6, 7], was criticized by Malmstrijm and Rosenberg [8] because direct measurements of bound metals were not made and the possibility that Mgz+ or Mnz+ might compete with another bound metal was not considered. These criticisms became important when it was found that preparations of leucine aminopeptidase contained Zn 2+ [9, 10]
Summary
Chemical Co. ZnO and MgSO,, used as spectroscopic standards, were the high purity salts from Johnson-Matthey and Co., Ltd. Trimethvlamine.HCI was from Eastman Organic Chemicals while N-ethvlmorpholine and 1.10.phenanthroline were obtained from Aldrich. Chemical Co. The N-ethylmorpholine was distilled before use. Crystalline leucine aminopeptidase was prepared from bovine lens according to the procedure of Hanson et al [20] as modified by Melbve [17]. All glass and plasticware was cleaned with concentrated HIC’OJH,SO, according to the method of Thiers [21]
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