INVOLVEMENT OF TRYPTOPHAN(S) AT THE ACTIVE-SITE OF POLYPHOSPHATE ATP GLUCOKINASE FROM MYCOBACTERIUM-TUBERCULOSIS
INVOLVEMENT OF TRYPTOPHAN(S) AT THE ACTIVE-SITE OF POLYPHOSPHATE ATP GLUCOKINASE FROM MYCOBACTERIUM-TUBERCULOSIS
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DOI：
10.1021/bi00075a018
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发表时间：
1993-06-22
期刊：
影响因子：
2.9
通讯作者：
PHILLIPS, NFB
中科院分区：
文献类型：
--
作者：
HSIEH, PC;SHENOY, BC;PHILLIPS, NFB
来自结核分枝杆菌的葡萄糖激酶 (EC2.7.1.63) 使用无机多磷酸盐 (poly(P)) 或 ATP 作为磷酰基供体来催化葡萄糖的磷酸化。通过使用 N-溴代琥珀酰亚胺 (NBS) 作为色氨酸参与底物结合和/或催化的探针，研究了聚 (P) 和 ATP 位点的性质。色氨酸的 NBS 氧化导致荧光猝灭，同时损失聚 (P) 和 ATP 依赖性葡萄糖激酶活性。 NBS 失活并不是由于广泛的结构变化，天然酶和 NBS 失活酶的相似圆二色光谱和荧光发射最大值证明了这一点。木糖存在下，两种磷酰基供体底物都能提供大约 65% 的保护，防止 NBS 失活。 NBS修饰后，poly(P)和ATP的K(m)值没有改变，但酶的催化效率降低，表明必需的色氨酸参与了底物的催化。丙烯酰胺猝灭研究表明色氨酸残基被底物部分屏蔽以防止猝灭。无配体葡萄糖激酶中色氨酸的 Stern-Volmer 猝灭常数 (K(SV)) 为 3.55 M-1，而在木糖+poly(P)5 和木糖+ATP 存在下分别获得 2.48 和 2.57 M-1 的 K(SV) 值。当通过肽图谱分析含色氨酸的肽时，发现木糖+聚(P)5和木糖+ATP保护相同的肽免受NBS氧化。通过N端序列分析和氨基酸组成确定这两个受保护的肽是相同的。从这些结果推测，受保护的肽中存在的色氨酸之一或两者可能位于共同的催化中心，并且该肽可能构成poly(P)和ATP结合区域的一部分。
The glucokinase (EC2.7.1.63) from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using inorganic polyphosphate (poly(P)) or ATP as the phosphoryl donor. The nature of the poly(P) and ATP sites was investigated by using N-bromosuccinimide (NBS) as a probe for the involvement of tryptophan in substrate binding and/or catalysis. NBS oxidation of the tryptophan(s) resulted in fluorescence quenching with concomitant loss of both the poly(P)- and ATP-dependent glucokinase activities. The inactivation by NBS was not due to extensive structural changes, as evidenced by similar circular dichroism spectra and fluorescence emission maxima for the native and NBS-inactivated enzyme. Both phosphoryl donor substrates in the presence of xylose afforded approximately 65% protection against inactivation by NBS. The K(m) values of poly(P) and ATP were not altered due to the modification by NBS, while the catalytic efficiency of the enzyme was decreased, suggesting that the essential tryptophan(s) are involved in the catalysis of the substrates. Acrylamide quenching studies indicated that the tryptophan residue(s) were partially shielded by the substrates against quenching. The Stern-Volmer quenching constant (K(SV)) of the tryptophans in unliganded glucokinase was 3.55 M-1, while K(SV) values of 2.48 and 2.57 M-1 were obtained in the presence of xylose+poly(P)5 and xylose+ATP, respectively. When the tryptophan-containing peptides were analyzed by peptide mapping, the same peptide was found to be protected by xylose+poly(P)5 and xylose+ATP against oxidation by NBS. The two protected peptides were determined to be identical by N-terminal sequence analysis and amino acid composition. It is proposed from these results that one or both of the tryptophans present in the protected peptide may be located at a common catalytic center and that this peptide may constitute part of the poly(P) and ATP binding regions.