人肌肌酸激酶胍变性时的失活与构象变化的比较研究

应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100％,则天然酶酪氨酸残基的暴露程度只有2％。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。

A COMPARISON OF CONFORMATIONAL CHANGES AND INACTIVATION RATES OF CREATINE KINASE FROM HUMAN MUSCLE DURING GUANIDINE DENATURATION

The conformational changes of creatine kinase from human muscle during denaturation by different concentrations of guanidine hydrochloride have been studied hy second derivative spectra, fluorescence and ultraviolet differential spectroscopic methods. The results obtained by second derivative spectra showed that if the hidden tyroune residues of the enzyme in 6M guanidine are considered to be exposed completely, only 2% tyrosine groups of native human creatine kinase are exposed to hydrophilic environment. The changes of fluorescence and UV differential spectra are the same as those of rabbit creatine kinase.Rate constants of conformational changes and inactivation of human creatine kinase during denaturation at the different concentrations of guanidine have been also determined. The conformational change courses in the different concentrations of guanidine have been to be first order reactions. However, inact-ivation are biphasic course. Comparison of rates of inactivation and conforma- tional changes show clearly that the rate constants of the fast phases of ina-ctivation of the human creatine kinase are 1-2 orders of magnitude faster than the rate constants of conformational changes, whereas slow phase constants are very close to the rate constants of conformational changes. Above results further support the suggestion of the location of the active site of the enzyme in limited and flexible molecular region.