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首页> 外文期刊>Biochemistry >Binding of Dioxygen to Non-Metal Sites in Proteins: Exploration of the Importance of Binding Site Size versus Hydrophobicity in the Copper Amine Oxidase from Hansenula polymorpha.
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Binding of Dioxygen to Non-Metal Sites in Proteins: Exploration of the Importance of Binding Site Size versus Hydrophobicity in the Copper Amine Oxidase from Hansenula polymorpha.

机译：氧与蛋白质中非金属位点的结合：探索多形汉逊酵母铜胺氧化酶中结合位点大小与疏水性的重要性。

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摘要

Copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and a copper ion in their active sites, catalyzing amine oxidation to aldehyde and ammonia concomitant with the reduction of molecular oxygen to hydrogen peroxide. Kinetic studies on the CAO from bovine serum (BSAO) [Su and Klinman (1999) Biochemistry 37, 12513-12525] and the recent reports on the cobalt substituted form of the enzyme from Hansenula polymorpha (HPAO) [Mills and Klinman (2000) J. Am. Chem. Soc. 122, 9897-9904, and Mills et al. (2002) Biochemistry, 41, 10577-10584] support pre-binding of molecular oxygen prior to a rate-limiting electron transfer from the reduced form of TPQ (p-aminohydroquinone form) to dioxygen. Although there is significant sequence homology between BSAO and HPAO, k(cat)/K(m)(O(2)) for BSAO under the optimal condition is one order of magnitude lower than that for HPAO. From a comparison of amino acid sequences for BSAO and HPAO, together with the X-ray crystal structure of HPAO, a plausible dioxygen pre-binding site has been identified that involves Y407, L425, and M634 in HPAO; the latter two residues are altered in BSAO to A490 and T695. To determine which of these residues plays a greater role in dioxygen chemistry, k(cat)/K(m)(O(2)) was determined in HPAO for the M634 --> T and L425 --> A mutants. The L425 --> A mutation does not alter k(cat)/K(m)(O(2)) to a large extent, whereas the M634 --> T decreased k(cat)/K(m)(O(2)) by one order of a magnitude, creating a catalyst that is similar to BSAO. A series of mutants at M634 (to F, L, and Q) were, therefore, prepared in HPAO and characterized with regard to k(cat)/K(m)(O(2)) as a function of pH. Structure reactivity correlations show a linear relationship of rate with side chain volume, rather than hydrophobicity, indicating that dioxygen reactivity increases with the bulk of the residue at position 634. This site also shows specificity for O(2), in relation to the co-gas N(2), since substitution of the inert gas N(2) by either Ar or He has no effect on measured rates. In particular, He gas is expected to have little affinity for protein at 1 atmospheric pressure, implying little or no binding by N(2) as well.
机译：铜胺氧化酶（CAO）在其活性位点中包含2,4,5-三羟基苯丙氨酰醌（TPQ）和铜离子，催化胺氧化为醛和氨，同时将分子氧还原为过氧化氢。牛血清中CAO的动力学研究[Su and Klinman（1999）Biochemistry 37，12513-12525]，以及多形汉逊酵母（HPAO）中酶的钴取代形式的最新报道[Mills and Klinman（2000） J.上午化学Soc。 122，9897-9904和Mills等人。 （2002）Biochemistry，41，10577-10584]支持在限速电子从还原形式的TPQ（对氨基氢醌形式）转移至双氧之前，分子氧的预结合。尽管BSAO和HPAO之间存在明显的序列同源性，但在最佳条件下BSAO的k（cat）/ K（m）（O（2））比HPAO的k（cat）/ K（m）（O（2））低一个数量级。通过比较BSAO和HPAO的氨基酸序列以及HPAO的X射线晶体结构，已经确定了一个合理的双氧预结合位点，该位点涉及HPAO中的Y407，L425和M634。后两个残基在BSAO中被改变为A490和T695。为了确定这些残基中的哪一个在双氧化学中发挥更大的作用，在HPAO中针对M634-> T和L425-> A突变体确定了k（cat）/ K（m）（O（2））。 L425->突变不会在很大程度上改变k（cat）/ K（m）（O（2）），而M634-> T降低了k（cat）/ K（m）（O（ 2））一个数量级，从而产生类似于BSAO的催化剂。因此，在HPAO中制备了一系列在M634（至F，L和Q）的突变体，并根据k（cat）/ K（m）（O（2））表征了pH的函数。结构反应性相关性显示速率与侧链体积呈线性关系，而不是疏水性，表明双氧反应性随位置634处的残基增加而增加。该位点还显示了对O（2）的特异性，相对于co-气体N（2），因为用Ar或He替代惰性气体N（2）对测得的速率没有影响。特别是，预计氦气在1个大气压下对蛋白质几乎没有亲和力，这意味着N（2）也几乎没有或没有结合。

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