组氨酸残基在乙醇酸氧化酶活性中的作用

DEPC能显著抑制GAO的活性。其失活速度表现为假一级动力学特性,并和抑制剂浓度成线性正比关系。底物乙醇酸可保护GAO免受DEPC抑制,羟胺能使被抑制的酶重新复活。光谱测定表明,被抑制的酶只有组氨酸残基被修饰,而酪氨酸残基未被修饰,修饰前后酶的氨基含量均无变化。反应动力学表明,在35℃下,GAO中有一个pK为6.5的解离基团和催化活性有关,其解离⊿H为31610 J/mol。因此组氨酸残基为GAO催化活性的一个必需基团。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅳ精氨酸残基在高粱叶片PEP羧化酶催化和调节功能中的作用

应用化学修饰的方法观察精氨酸残基在PEP羧化酶的催化和调节功能中的作用。用丁二酮在硼酸盐缓冲液存在下处理PEP羧化酶使酶活性迅速丧失。其失活速度表现为拟一级反应动力学特性。 低温处理(15℃),或者PEP、G6P、甘氨酸,苹果酸,G6P加甘氨酸和PEP加甘氨酸等酶的底物和效应剂的存在对酶的丁二酮失活均具不同程度的保护作用。PEP和G6P的P_(0.5)值各为4mM和1.5mM。 丁二酮对酶的修饰表现为可逆失活。在Tris-H_2SO_4缓冲液中透析可使被丁二酮修饰而丧失的酶活性恢复。 丁二酮处理还使酶失去对G6P的敏感性,但不影响甘氨酸对酶的调节作用。低温(15℃)下丁二酮修饰酶的G6P脱敏速度比常温下(30℃)底物保护的修饰酶的G6P脱敏速度慢。比较脱敏速度常数(k_(dG6P))前者是0.0116(分~(-1)),后者是0.0562(分~(-1))。甘氨酸的加入不影响底物保护的修饰酶的G6P脱敏速度而明显降低酶的丁二酮失活速度。 这些结果表明精氨酸残基不仅存在于酶的催化部位并为酶的催化所必需,同时还存在于酶的G6P结合部位而参与G6P对酶的调节功能。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅶ.PEP羧化酶必需赖氨酸残基的化学修饰

本文报道纯化的高粱叶片PEP 羧化酶经氨基修饰剂TNBS 和PLP 的修饰迅速失活。酶的TNBS 失活与保温时间和抑制剂浓度呈函数关系并表现为拟一级反应的特性。动力学资料表明酶仅被1分子TNBS 修饰即失活。TNBS 修饰酶的吸收光谱特性表明被修饰的是酶蛋白的赖氨酸残基。底物(PEP)和效应剂(G6P)保护酶免被TNBS 失活。计算G6P 和酶的解离常数K_d-2.39×10~(-3)M。酶的其他反应组分HCO_3~-和MgCl_2单独存在时均不影响TNBS 对酶的失活作用。在被TNBS 修饰过程中还导致酶对G6P 迅速脱敏,同时却保持酶对甘氨酸的敏感性。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅶ.PEP羧化酶必需赖氨酸残基的化学修饰

本文报道纯化的高粱叶片PEP羧化酶经氨基修饰剂TNBS和PLP的修饰迅速失活。酶的TNBS失活与保温时间和抑制剂浓度呈函数关系并表现为拟一级反应的特性。动力学资料表明酶仅被1分子TNBS修饰即失活。TNBS修饰酶的吸收光谱特性表明被修饰的是酶蛋白的赖氨酸残基。底物(PEP)和效应剂(G6P)保护酶免被TNBS失活。计算G6P和酶的解离常数K_d=2.39×10~(-3)M。酶的其他反应组分HCO_3~-和MgCl_2单独存在时均不影响TNBS对酶的失活作用。在被TNBS修饰过程中还导致酶对G6P迅速脱敏,同时却保持酶对甘氨酸的敏感性。     

兔肌甘油醛-3-磷酸脱氢酶中NAD~ 类似物对碘乙酸修饰作用及酶促乙酰磷酸水解作用的影响

碘乙酸修饰兔肌甘油醛-3-磷酸脱氢酶蛋白过程中,以NAD~ 类似物代替NAD~ 测定它们对酶蛋白失活速度的影响。在被测的五个NAD~ 类似物中CPAD~ ,FPAD~ 对酶有保护作用,降低了酶因修饰而失活的速度。其它三个类似物NGD~ ,APAD~ 和TPAD~ 在不同程度上加速碘乙酸引起的失活。这些类似物在对碘乙酸修饰速度造成影响方面表现的能力与它们作为氢受体能力相一致。以NAD~ 类似物代替NAD~ 测定它们对酶促乙酰磷酸水解作用的影响。与碘乙酸修饰时有些不同,CPAD~ ,TPAD~ 对这一水解过程无促进作用,而FPAD~ ,NGD~ 和APAD~ 有促进作用。各类似物的行为与羧甲基酶光照形成新萤光团过程中各类似物的行为相一致。     

兔肌甘油醛-3-磷酸脱氢酶中NAD~ 类似物对碘乙酸修饰作用及酶促乙酰磷酸水解作用的影响

碘乙酸修饰兔肌甘油醛-3-磷酸脱氨酶蛋白过程中,以NAD~ 类似物代替NAD~ 测定它们对酶蛋白失活速度的影响。在被测的五个NAD~ 类似物中CPAD~ ,FPAD~ 对酶有保护作用,降低了酶因修饰而失活的速度。其它三个类似物NGD~ ,APAD~ 和TPAD~ 在不同程度上加速碘乙酸引起的失活。这些类似物在对碘乙酸修饰速度造成影响方面表现的能力与它们作为氢受体能力相一致。以NAD~ 类似物代替NAD~ 测定它们对酶促乙酰磷酸水解作用的影响。与碘乙酸修饰时有些不同,CPAD~ ,TPAD~ 对这一水解过程无促进作用,而FPAD~ ,NGD~ 和APAD~ 有促进作用。各类似物的行为与羧甲基酶光照形成新萤光团过程中各类似物的行为相一致。     

乙醇酸氧化酶的纯化结晶和酪氨酸残基的修饰对酶活性的影响

从青菜叶片中纯化的GAO为淡黄色棒状或针状结晶,纯化度达129.3倍。NBS对GAO有强烈的抑制作用,其抑制效应不受pH的影响,抑制程度和 NBS浓度(0～10 μmol/L)呈线性关系;乙醇酸和草酸均能保护GAO免受NBS的抑制。NBS在全抑制浓度范围内并不降低OD_(280),但引起OD_(260)明显上升;当用乙醇酸保护GAO时,这种上升与酶的抑制同时消失。另外两种更专一的酪氨酸残基修饰剂NBSF和N—AI亦显著抑制GAO活性,底物乙醇酸对此有保护作用,N—AI抑制的GAO可被羟胺复活。     

人胎盘谷胱甘肽S-转移酶中巯基的研究

 用巯基试剂5.5'-二硫双(2-硝基苯甲酸)(DTNB)测得人胎盘谷胱甘肽S-转移酶(GST-π)的总巯基数为每亚基2个,均为表面巯基,,其中一个与DTNB反应快,被修饰后可导致酶活力全部丧失。另一巯基与DTNB反应较慢,可能与酶活力无关。用在12℃测定剩余巯基和Stallcup-Koshland作图法求得DTNB修饰快反应和慢反应巯基的速度常数分别为44056和162min~(-1)(mol/L)~(-1)。底物谷胱甘肽的衍生物S-正辛烷谷胱甘肽(S-o-GSH)能保护GST-π能保护的快反应巯基免受DTNB的修饰,使反应速度常数随着S-o-GSH浓度的增高而降低。S-o-GSH也能保护酶被N-乙基马来酰亚胺(NEMI)修饰失活,但不能保护慢反应巯基被DTNB修饰。另一底物2,4-二硝基氯苯(CDNB)对NEMI的修饰失活没有保护作用。上述结果提示快反应巯基参与GST-π和谷胱甘肽的结合,是组成活性中心的重要基因。     

化学修饰对胰激肽释放酶稳定性及其性质的影响

以自制高活性ＰＰＫ为材料,在氰基硼氢化钠存在下经水溶性乙醛酸修饰其表面氨基,使其抗不可逆热失活稳定性有显著提高。结果表明,修饰ＰＰＫ在等电点等基本性质方面都有变化。修饰ＰＰＫ的ＢＡＥＥ活性为天然酶的８２％,氨基修饰度为５８％,抗蛋白酶水解,贮藏和冻干稳定性都有加强。     

谷胱甘肽S-转移酶活性中心的研究—羧基、氨基和胍基的化学修饰

用1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺(EDC),2.4.6.三硝基苯磺酸(TNBS)和丁二酮(DIC)分别修饰人胎盘型谷胱甘肽S-转移酶(GST-π)的羧基、氨基和胍基,研究了酶的失活动力学,发现引起一分子酶亚基全部失活所需抑制剂的分子数分别为1.0、1.08和0.98,提示每亚基只有一个羧基、氨基和胍基参与酶的活性中心。底物及其类似物谷胱甘肽,S-己烷或S-辛烷谷胱甘肽可保护GST-π免受上述抑制剂的修饰,使假一级反应速度常数k_1明显降低,说明羧基、氨基和胍基是GST-π和GSH结合部位的组成基团。作者曾证明GST-π中的一个快反应巯基也参与酶与GSH的结合,故至少有四个不同的基团是酶亚基的结合基团,本文还对TNBS对氨基修饰的特异性作了验证,并讨论了GST-π与GSH结合时形成离子键的情况。     

Phosphoglycerate mutase has essential arginyl residues

Phosphoglycerate mutase is inactivated by butanedione in borate buffer. Inactivation by 0.13 mM reagent correlates with the modification of one arginyl residue per subunit, and is prevented by either 2, 3-diphosphoglycerate or 3-phosphoglycerate. With 0.50 mM butanedione, inactivation is accompanied by the modification of three arginyl residues per subunit, two of which are protected by the combined presence of cofactor and substrate.     

Essential arginyl residues in yeast enolase.

Yeast enolase is rapidly inactivated by butanedione in borate buffer, complete inactivation correlating with the modification of 1. 8 arginyl residues per subunit. Protection against inactivation is provided by either an equilibrium mixture of substrates or inorganic phosphate, a competitive inhibitor of the enzyme. Complete protection by substrates correlates with the shielding of 1. 3 arginyl residues per subunit, while phosphate protects 1. 0 arginyl residue per subunit from modification.     

Evidence of essential arginyl residues in rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase is inactivated by 2,3-butanedione in borate buffer. The inactivation follows pseudo-first-order kinetics with a calculated second-order rate constant of 4.6 m^?1 min^?1. The modification can be reversed with almost total recovery of activity by elimination of the butanedione and borate buffer, suggesting that only arginyl groups are modified; this result agrees with the loss of arginine detected by amino acid analysis of the modified enzyme. Using the kinetic data, it was estimated that the reaction of a single butanedione molecule per subunit of the enzyme is enough to completely inactivate the protein. The inactivation is partially prevented by phosphoenolpyruvate in the presence of K⁺ and Mg²⁺, but not by the competitive inhibitors lactate and bicarbonate. These findings point to an essential arginyl residue being located near the phosphate binding site of phosphoenolpyruvate.     

Identification of essential arginyl residues in cytoplasmic malate dehydrogenase with butanedione.

The inactivation of cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) from porcine heart and the specific modification of arginyl residues have been found to occur when the enzyme is inhibited with the reagent butanedione in sodium borate buffer. The inactivation of the enzyme was found to follow pseudo-first order kinetics. This loss of enzymatic activity was concomitant with the modification of 4 arginyl residues per molecule of enzyme. All 4 residues could be made inaccessible to modification when a malate dehydrogenase-NADH-hydroxymalonate ternary complex was formed. Only 2 of the residues were protected by NADH alone and appear to be essential. Studies of the butanedione inactivation in sodium phosphate buffer and of reactivation of enzymatic activity, upon the removal of excess butanedione and borate, support the role of borate ion stabilization in the inactivation mechanism previously reported by Riordan (Riordan, J.F. (1970) Fed. Proc. 29, Abstr. 462; Riordan, J.F. (1973) Biochemistry 12, 3915-3923). Protection from inactivation was also provided by the competitive inhibitor AMP, while nicotinamide exhibited no effect. Such results suggest that the AMP moiety of the NADH molecule is of major importance in the ability of NADH to protect the enzyme. When fluorescence titrations were used to monitor the ability of cytoplasmic malate dehydrogenase to form a binary complex with NADH and to form a ternary complex with NADH and hydroxymalonate, only the formation of ternary complex seemed to be effected by arginine modification.     

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.     

Essential arginyl residues in fructose-1,6-bisphosphatase.

Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.     

Evidence for an essential arginyl residue in bovine milk gamma-glutamyltransferase

Treatment of bovine milk gamma-glutamyltransferase with 2,3-butanedione in borate buffer markedly inactivates its gamma-glutamyltransferase activity. Inactivation is prevented by a combination of the gamma-glutamyl donor and acceptor substrates, glutathione, and glycylglycine, but less effectively by only one of them. Serine plus borate of maleate provides no protection against the inactivation. Amino acid analysis of the enzyme treated with butanedione in the presence and absence of the protecting substrate combination indicates that complete inactivation correlates with the modification of a single arginyl residue per molecule. The residue modified is associated with the smaller subunit of the two equal subunits which comprise the enzyme. The butanedione-treated enzyme retains a hydrolytic activity, another but less significant catalytic function of the enzyme. The results indicate that the arginyl residue is involved in recognizing the anionic moiety of the acceptor and in binding it to the acceptor site located on the smaller subunit of the enzyme.     

The role of arginine residues in the function of D-glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of apo-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle in the presence of butanedione is the result of modification of one arginyl residue per subunit of the tetrameric enzyme molecule. The loss of activity follows pseudo-first-order kinetics. NAD+ increases the apparent first-order rate constant of inactivation. The effect of NAD+ on the enzyme inactivation is cooperative (Hill coefficient = 2.3--3.2). Glyceraldehyde 3-phosphate protected the holoenzyme against inactivation, decreasing the rate constant of the reaction. At saturating concentrations of substrate the protection was complete. The Hill plot demonstrates that the effect is cooperative. This suggests that subunit interactions in the tetrameric holoenzyme molecule may affect the reactivity of the essential arginyl residues. In contrast, glyceraldehyde 3-phosphate had no effect on the rate of inactivation of the apoenzyme in the presence of butanedione. 100 mM inorganic phosphate protected both the apoenzyme and holoenzyme against inactivation. The involvement of the microenvironment of the arginyl residues in the functionally important conformational changes of the enzyme is discussed.     

Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.     

An essential arginine residue in the ATP-binding centre of (Na+ + K+)-ATPase.

1. Incubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rabbit kidney outer medulla with butanedione in borate buffer leads to reversible inactivation of the (Na+ + K+)-ATPase activity. 2. The reaction shows second-outer kinetics, suggesting that modification of a single amino acid residue is involved in the inactivation of the enzyme. 3. The pH dependence of the reaction and the effect of borate ions strongly suggest that modification of an arginine residue is involved. 4. Replacement of Na+ by K+ in the butanedione medium decreases inactivation. 5. ATP, ADP and adenylyl imido diphosphate, particularly in the presence of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid to complex Mg2+, protect the enzyme very efficiently against inactivation by butanedione. 6. The (Na+ + Mg2+)-dependent phosphorylation capacity of the enzyme is inhibited in the same degree as the (Na+ + K+)-ATPase activity by butanedione. 7. The K+-stimulated p-nitrophenylphosphatase activity is much less inhibited than the (Na+ + K+)ATPase activity. 8. The ATP stimulation of the K+-stimulated p-nitrophenylphosphatase activity is inhibited by butanedione to the same extent as the (Na+ + K+)-ATPase activity. 9. Modification of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) protects partially against the inactivating effect of butanedione. 10. The results suggest that an arginine residue is present in the nucleotide binding centre of the enzyme.