产碱杆菌Alcaligenes sp.KS-85来源肌酸酶活性中心的关键氨基酸功能研究

肌酸酶(Creatinase,EC 3.5.3.3)水解肌酸生成尿素和肌氨酸,是肌酐多酶级联检测中的关键酶。为进一步解析产碱杆菌来源肌酸酶的催化机理,利用蛋白质同源建模、分子对接、丙氨酸扫描技术分析了酶与底物的相互作用,并聚焦于酶活性中心4个功能未知的保守位点Phe64、Asp102、Phe252、Phe321,通过将氨基酸残基定点突变为6种具有代表性的氨基酸,结合生化实验对其功能进行解析。经研究,所有突变体的kcat值大幅度降低;除6个突变体:F252A、F252S、F252Y、F252W、F321A、F321Y的KM值降低,其余突变体的KM均提高。结构分析表明Phe252通过与Tyr259形成的π-π堆积稳定酶-底物复合体;Asp102通过与Arg66,Gly322的氢键相互作用稳定酶反应的过渡态;Phe321,Phe64位于底物两侧,通过疏水侧链的排斥作用及空间位阻影响底物的定位。本研究通过对活性中心氨基酸残基的功能解析,为肌酸酶的催化机制解析和分子改造奠定了基础。     

Q20L及G247D定点突变对葡萄糖异构酶酶活和最适pH的改善

用双引物法对GI基因进行体外定点突变,构建了突变体Q20L和G247D。含突变基因的重组表达质粒pTKD-GIQ20L及pTKDGIG247D在E.coli K38菌株中表达。纯化的突变酶与野生型酶相比：(1) GIQ20L的最适反应温度下降5℃,热稳定性为野生型酶的78%,对底物的亲和性增强;(2) GIG247D的酶活提高约33%,最适pH下降0.6个单位,但热稳定性降低。初步分析认为,Gln 20位于α0～α1螺旋之间,其亲水侧链被Leu的疏水侧链取代后,分子表面增强的疏水作用,反而不利于蛋白质的稳定,使GIQ20L的热稳定性降低。Gly247是酶活性中心β折叠(242～247aa)的最后一个残基。引入电负性极强的Asp后,可能改变分子的静电场分布,影响了活性部位的电荷传递过程,使GIG247D酶活提高。引入的电荷,可能改变活性中心可解离基团的pKa,使其最适pH下降。另外Asp247的侧链在周围空间结构中显得过于拥挤,易与其他侧链产生排斥,由此影响到β-折叠的稳定性,接近亚基结合面的Asp247,可能进一步影响到亚基间相互作用的稳定性,最终导致酶热稳定性的降低。GI酶活和最适pH的改善更利于工业生产。     

定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶,其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成,并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变,通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用,通过Ni2+- NTA亲和层析对D165及N192突变子进行纯化,对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%,E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达,表达量分别为野生型的28.2%和20.8%,突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274,其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。     

抗真菌药物作用靶酶羊毛甾醇14α去甲基化酶研究

羊毛甾醇14α去甲基化酶是普遍存在于高等植物、真菌和哺乳动物体内的P450蛋白,是氮唑类抗真菌药物作用靶酶.到目前为止已分别确定了高等植物、真菌和哺乳动物体内该酶的氨基酸序列.该酶对底物的催化包括三个单加氧步骤,涉及自由基的生成和消除,血红素辅基在酶催化过程中起重要作用.底物羊毛甾醇只能结合在酶活性位点血红素辅基Nc吡咯环上方,其余血红素吡咯环被氨基酸残基封闭.底物羊毛甾醇的3β羟基、Δ⁸⁽⁹⁾双键和17位侧链是与酶活性位点正确结合的关键官能团.该酶两大类抑制剂(底物类似物和氮唑类抗真菌药物)结构-活性关系研究可为进一步优化和设计新型高效酶抑制剂提供基础.     

R-2-氯丙酸脱卤酶的定向进化

通过易错PCR手段将R-2-氯丙酸脱卤酶定向进化,并使用基于Cl-浓度显色反应的高通量筛选得到有效突变子库,发现突变子DehDIV-G2和DehDIV-E7的酶比活力分别提高25.2%和38.7%。通过SYBYL对酶与底物进行分子对接显示,DehDIV-G2的活化能下降0.961 4 kJ/mol,DehDIV-E7的活化能下降2.549 8 kJ/mol。由于酶和底物R-2-氯丙酸的活化能下降,亲和能力提高,从而提高酶的比活力。     

快速展示糖苷水解酶活性架构单点突变导致结合力变化的电泳技术

酶分子在长期进化过程中形成一系列氨基酸残基组成的活性架构,参与底物的识别、结合与催化过程,而活性架构中相应氨基酸残基是如何影响酶分子结合底物的能力,进而影响酶分子的催化效率,一直是酶分子理性改造研究的热点.利用亲和电泳技术,可以快速展示内切纤维素酶Tr Cel12A和木聚糖酶Tl Xyn A活性架构中不同突变体的催化活性及其迁移率的变化,进而通过在不同底物浓度凝胶中蛋白质相对迁移率变化程度的定量回归分析,发现由氨基酸单点突变导致蛋白质迁移率的相对变化,可以定量表征酶分子突变前后结合底物能力的变化.亲和电泳测定的有效阻滞常数Kb值与等温滴定量热法和荧光光谱法测定的相关参数比较具有明显相关性.由于亲和电泳技术在测定酶分子与底物的结合能力时具有简便、快速、灵敏的特点,因而可作为常规生化实验室常规普筛技术来检测突变文库中系列突变体导致结合力的变化.     

腾冲嗜酸热两面菌伴侣素ATcpnβ底物结合特性研究

嗜酸嗜热古菌腾冲嗜酸热两面菌(Acidianus tengchongensis)来源的Ⅱ型伴侣素ATcpnβ已获得晶体结构解析,其顶端结构域突触下端相应于Ⅰ型伴侣素GroEL的重要底物结合位点处的氨基酸多为极性氨基酸,将其突变为疏水性氨基酸时,突变体对变性底物的捕获能力显著增强.表面等离子共振研究表明,ATcpnβ对于化学变性底物的再折叠中间体的其捕获作用不依赖于Mg2+/ATP.前期对该伴侣素冷冻电镜观察和结构解析表明,ATP的存在并不能驱动ATcpnβ从开放构型向闭合构型转变,但是表面等离子共振研究表明,ATcpnβ对热变性过程中已经聚集的底物的捕获作用依赖于Mg2+/ATP,说明Mg2+/ATP可以介导ATcpnβ顶端结构域一定的构象变化,引起顶端结构域疏水残基的进一步暴露,从而能够与大分子聚集体紧密结合.两个方面的研究均表明,伴侣素蛋白与变性底物的结合仍然以疏水相互作用为主,并且伴侣素蛋白与变性底物的结合受Mg2+/ATP的结合调控,与伴侣素蛋白疏水面的暴露程度相关.     

花青素主要成分与HER-2激酶区的分子对接

用分子对接方法预测天然植物化学物质与受体蛋白的相互作用位点并探究作用机制。利用MVD(Molecular Virtual Docker 5.5)软件,以HER-2激酶区为受体模板建立活性位点,与12种花青素成分进行分子对接。结果表明12种化合物均能在同一活性腔中与HER-2激酶区对接(MolDock Score:苷元–105 kJ/mol,单葡糖苷–130 kJ/mol),主要作用力是疏水作用和氢键;该活性腔也是ATP与HER-2激酶区的结合(MolDock Score=–161 kJ/mol)位点,花青素的结合可能会干扰ATP与HER-2之间氢键的形成。提示花青素可能以竞争性结合方式阻碍ATP与HER-2的结合,抑制HER-2磷酸化激活及下游信号通路的激活,从而发挥抑癌活性。     

定点突变改变近平滑假丝酵母SCRⅡ催化苯乙酮衍生物底物谱

【目的】通过定点突变技术,改变近平滑假丝酵母短链羰基还原酶Ⅱ(SCRⅡ)催化苯乙酮衍生物的功能,为数种手性芳香醇的生产提供一种高效、安全的新型制备方法。【方法】通过氨基酸序列和蛋白结构比对的方法,选择SCRⅡ的底物结合域中关键氨基酸位点E228实施突变,构建相应的突变株Escherichia coliBL21/pET28a-E228S;以苯乙酮衍生物为底物,对突变株的酶活和生物转化功能进行了分析。【结果】酶活测定结果表明:突变株E.coli BL21/pET28a-E228S催化原始底物2-羟基苯乙酮的酶活仅为原始酶活的25%左右;而催化苯乙酮、4’-甲基苯乙酮、4’-氯苯乙酮的酶活是突变前的7-20倍。突变株E.coli BL21/pET28a-E228S生物转化2-羟基苯乙酮,获得产物(S)-苯基乙二醇的得率不超过10%,而以苯乙酮、4’-甲基苯乙酮、4’-氯苯乙酮为底物时,生物转化产物光学纯度维持在99%,得率高达80%以上。【结论】对底物结合域中的关键氨基酸实施突变,提高了SCRⅡ催化苯乙酮衍生物的底物广谱性,拓展了该酶的生物功能,为理性改造短链羰基还原酶的不对称还原催化功能和手性芳香醇的制备提供了新型途径。     

α-环糊精糖基转移酶活性区域突变提高选择形成γ-环糊精能力

探讨α-环糊精糖基转移酶(CGT酶)活性区域-3亚位点(47位赖氨酸残基),-7亚位点(146~152位氨基酸残基)以及环化中心位点(195位酪氨酸残基)对其催化底物形成γ-环糊精(CD)能力的影响。将α-CGT酶相应位点分别进行如下突变:K47T,Y195I,以及146~152位氨基酸残基替换为异亮氨酸(命名为△6),并在大肠杆菌BL21中实现异源活性表达。以可溶性淀粉作为底物进行转化,利用HPLC分析各种突变酶的催化产物中3种环糊精产量和比例。结果表明,和野生酶相比,所有突变酶的淀粉水解活性和环糊精总生成量都有不同程度的下降。在产物的组成方面,突变酶Y195I的催化产物中,α-CD的含量由68%降为30%,β-CD由22.2%提高为33.3%;而γ-CD由8.9%提高为36.7%,含量提高了4倍,取代α-CD成为产物中的主要成分;γ-CD的实际产量为1.1 g/L,是野生酶(0.4 g/L)的3倍。突变酶K47T和△6的转化产物中α-CD比例有不同程度下降,但仍然是产物中的主要组分,β-和γ-CD的比例都有所增加。由此可见,活性区域中195位氨基酸对于α-CGT酶的活力和催化选择性具有重要的影响,Y195I突变体酶最有利于选择性形成γ-CD。纯化后突变酶Y195I的酶学性质试验表明,其最适反应温度和野生酶相同,但最适反应pH有所提高,且比野生酶具有更好的pH稳定性。因此,突变酶Y195I具有生产制备γ-CD的潜力。     

Hydrophobic nature of the active site of mandelate racemase

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, stabilizing the altered substrate in the transition state by approximately 26 kcal/mol. We have used a series of substrate analogues (glycolates) and intermediate analogues (hydroxamates) to evaluate the contribution of the hydrophobic cavity within the enzyme's active site to ligand binding. Free energy changes accompanying binding of glycolate derivatives correlated well with the hydrophobic substituent constant pi and the van der Waals surface areas of the ligands. The observed dependence of the apparent binding free energy on surface area of the ligand was -30 +/- 5 cal mol(-1) A(-2) at 25 degrees C. Free energy changes accompanying binding of hydroxamate derivatives also correlated well with pi values and the van der Waals surface areas of the ligands, giving a slightly greater free energy dependence equal to -41 +/- 3 cal mol(-1) A(-2) at 25 degrees C. Surprisingly, mandelate racemase exhibited a binding affinity for the intermediate analogue benzohydroxamate that was 2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions. This suggests that there are additional specific interactions that stabilize the altered substrate in the transition state. Mandelate racemase was competitively inhibited by (R,S)-1-naphthylglycolate (apparent K(i) = 1.9 +/- 0.1 mM) and (R,S)-2-naphthylglycolate (apparent K(i) = 0.52 +/- 0.03 mM), demonstrating the plasticity of the hydrophobic pocket. Both (R)- (K(m) = 0.46 +/- 0.06 mM, k(cat) = 33 +/- 1 s(-1)) and (S)-2-naphthylglycolate (K(m) = 0.41 +/- 0.03 mM, k(cat) = 25 +/- 1 s(-1)) were shown to be alternative substrates for mandelate racemase. These kinetic results demonstrate that no major steric restrictions are imposed on the binding of this bulkier substrate in the ground state but that steric factors appear to impair transition state/intermediate stabilization. 2-Naphthohydroxamate was identified as a competitive inhibitor of mandelate racemase, binding with an affinity (K(i) = 57 +/- 18 microM) that was reduced relative to that observed for benzohydroxamate and that was in accord with the approximately 10-fold reduction in the value of k(cat)/K(m) for the racemization of 2-naphthylglycolate. These findings indicate that, for mandelate racemase, steric constraints within the hydrophobic cavity of the enzyme-intermediate complex are more stringent than those in the enzyme-substrate complex.     

Mutational analysis of the active site flap (20s loop) of mandelate racemase

Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG with kcat/Km values approximately 2-fold greater than those observed for wild-type mandelate racemase. Although minor changes in substrate specificity were achieved through alterations of the active site flap of mandelate racemase, our results suggest that hydrophobic residues that reside on a flexible flap and define the topology of an active site through their van der Waals contacts with the substrate are quite tolerant of a variety of steric substitutions.     

The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction

Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain alpha-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40 degrees. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with beta-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby beta-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.     

Membrane binding of the colicin E1 channel: activity requires an electrostatic interaction of intermediate magnitude.

In vitro channel activity of the C-terminal colicin E1 channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, I = 0.12 M, pH 4.0), the free energy of binding was delta G approximately -9 kcal/mol, with nonelectrostatic and electrostatic components, delta Gnel approximately -5 kcal/mol and delta Gel approximately -4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.     

Dissecting the Forces that Dominate Dimerization of the Nucleotide Binding Domains of ABCB1

P-glycoprotein, also known as multidrug resistance protein 1 or ABCB1, can export a wide range of chemically unrelated compounds, including chemotherapeutic drugs. ABCB1 consists of two transmembrane domains that form the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that energize substrate transport by ATP binding and hydrolysis. ATP binding triggers dimerization of the NBDs, which switches the transporter from an inward facing to an outward facing transmembrane domain conformation. We performed MD simulations to study the dynamic behavior of the NBD dimer in the presence or absence of nucleotides. In the apo configuration, the NBDs were overall attractive to each other as shown in the potential of mean force profile, but the energy well was shallow and broad. In contrast, a sharp and deep energy minimum (～?42 kJ/mol) was found in the presence of ATP, leading to a well-defined conformation. Motif interaction network analyses revealed that ATP stabilizes the NBD dimer by serving as the central hub for interdomain connections. Simulations showed that forces promoting dimerization are multilayered, dominated by electrostatic interactions between the nucleotide and conserved amino acids of the signature sequence and the Walker A motif. In addition, direct and water-bridged hydrogen bonds between NBDs provided conformation-defining interactions. Importantly, we characterized a largely unrecognized but essential contribution from hydrophobic interactions between the adenine moiety of the nucleotides and a hydrophobic surface of the X-loop to the stabilization of the nucleotide-bound NBD dimer. These hydrophobic interactions lead to a sharp energy minimum, thereby conformationally restricting the nucleotide-bound state.     

Electrostatic stabilization of a thermophilic cold shock protein.

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket.

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T1.

Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.     

The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.