Erman J E, Vitello L B, Miller M A, Shaw A, Brown K A, Kraut J
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Biochemistry. 1993 Sep 21;32(37):9798-806. doi: 10.1021/bi00088a035.
The crystal structure and reactivity with hydrogen peroxide are reported for a mutant of a cloned cytochrome c peroxidase [CcP(MI)], in which the conserved distal His (His-52) is replaced with Leu. The reaction of the H52L enzyme with peroxide was examined as a function of pH in 0.1 M phosphate buffers and buffers in which nitrate was used to adjust the ionic strength. The pH-independent bimolecular rate constant for the reaction of H52L with peroxide was 731 +/- 44 and 236 +/- 14 M-1 s-1 in phosphate and nitrate-containing buffers, respectively. This represents a 10(5)-fold decrease in rate relative to the CcP(MI) parent under comparable conditions. Single-crystal diffraction studies showed that no dramatic changes in the structure or in the accessibility of the heme binding site were caused by the mutation. Rather, the mutation caused significant structural changes only at residue 52 and the nearby active-site water molecules. The residual reactivity of the H52L enzyme with peroxide was pH- and buffer-dependent. In nitrate-containing buffer, the apparent bimolecular rate constant for the reaction with peroxide decreased with decreasing pH; the loss of reactivity correlated with protonation of a group with an apparent pKA = 4.5. Protonation of the group caused a loss of reactivity with peroxide. This is in contrast to the CcP(MI) parent enzyme, as well as all other mutants that have been examined, where the loss of reactivity correlates with protonation of an enzyme group with an apparent pKA = 5.4.(ABSTRACT TRUNCATED AT 250 WORDS)
本文报道了克隆的细胞色素c过氧化物酶[CcP(MI)]的一个突变体的晶体结构及其与过氧化氢的反应活性，该突变体中保守的远端组氨酸（His-52）被亮氨酸取代。在0.1 M磷酸盐缓冲液以及用硝酸盐调节离子强度的缓冲液中，研究了H52L酶与过氧化物的反应随pH的变化。在磷酸盐缓冲液和含硝酸盐的缓冲液中，H52L与过氧化物反应的pH无关双分子速率常数分别为731±44和236±14 M-1 s-1。这表明在可比条件下，相对于CcP(MI)亲本，反应速率下降了10^5倍。单晶衍射研究表明，该突变未导致结构或血红素结合位点可及性的显著变化。相反，该突变仅在52位残基和附近的活性位点水分子处引起了显著的结构变化。H52L酶与过氧化物的残余反应活性与pH和缓冲液有关。在含硝酸盐的缓冲液中，与过氧化物反应的表观双分子速率常数随pH降低而降低；反应活性的丧失与表观pKA = 4.5的基团的质子化相关。该基团的质子化导致与过氧化物反应活性的丧失。这与CcP(MI)亲本酶以及所有其他已研究的突变体形成对比，在那些突变体中，反应活性的丧失与表观pKA = 5.4的酶基团的质子化相关。（摘要截短于250字）
