Hsieh P C, Shenoy B C, Haase F C, Jentoft J E, Phillips N F
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Biochemistry. 1993 Jun 22;32(24):6243-9. doi: 10.1021/bi00075a018.
The glucokinase (EC 2.7.1.63) 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 Km 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 (KSV) of the tryptophans in unliganded glucokinase was 3.55 M-1, while KSV 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.(ABSTRACT TRUNCATED AT 250 WORDS)
结核分枝杆菌的葡萄糖激酶（EC 2.7.1.63）以无机多聚磷酸盐（多聚（P））或ATP作为磷酰基供体催化葡萄糖磷酸化。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/或催化作用的探针，研究了多聚（P）和ATP位点的性质。色氨酸的NBS氧化导致荧光猝灭，同时多聚（P）依赖性和ATP依赖性葡萄糖激酶活性丧失。NBS引起的失活并非由于广泛的结构变化，天然酶和NBS失活酶的圆二色光谱和荧光发射最大值相似证明了这一点。在木糖存在下，两种磷酰基供体底物均可提供约65%的保护，防止被NBS失活。多聚（P）和ATP的Km值并未因NBS修饰而改变，而酶的催化效率降低，这表明必需的色氨酸参与底物的催化作用。丙烯酰胺猝灭研究表明，色氨酸残基被底物部分屏蔽，防止猝灭。未结合配体的葡萄糖激酶中色氨酸的Stern-Volmer猝灭常数（KSV）为3.55 M-1，而在木糖+多聚（P）5和木糖+ATP存在下，KSV值分别为2.48和2.57 M-1。当通过肽图谱分析含色氨酸的肽时，发现相同的肽被木糖+多聚（P）5和木糖+ATP保护，免受NBS氧化。通过N端序列分析和氨基酸组成确定这两种受保护的肽是相同的。（摘要截短至250字）
