用戶:Inzhrui/沙盒4

酶催化混雜（enzyme promiscuity）是指一種酶除了主要反應外，還能偶然催化一種副反應。儘管酶是專一性強的催化劑，但它們除了具有主要的天然催化活性外，還經常會發生副反應。這些混雜的反應通常比主要反應慢，在中性選擇下。儘管這些反應在生理上通常是不相關的，但在新的自然選擇壓力下，這些活動可能會帶來適應效益，從而促使以前的混雜反應演變成新的主要反應。 酶催化混雜的一個例子是來自假單胞菌屬ADP的阿特拉津氯水解酶（編碼atzA），其由三聚氰胺脫氨酶（編碼triA）演化而來，其對人造化學品阿特拉津具有非常小的混雜活性。

介紹

酶進化為在特定底物上以高催化效率（k_cat/K_M，參見米-門二氏動力學）催化特定反應的催化劑。然而，除了這一主要活性外，它們還具有其他活性，這些活性通常要低幾個數量級，並且不是進化選擇的結果，因此不參與生物體的生理活動。 該現象允許新功能的獲得，因為混雜的活性可以在新的選擇壓力下賦予適應效益，導致其作為新的主要活性的重複和選擇。

酶的進化

重複和專門化

有幾種理論模型可以預測重複和專門化事件的順序，但實際過程更為複雜和模糊（§下文重構酶）。 一方面，基因擴增導致酶濃度增加，並且可能不受限制性調節，因此增加了酶混雜活性的反應速率（v），使其作用在生理上更加明顯（「基因劑量效應」）。 另一方面，酶可能會增加次要活性，而幾乎沒有損失主要活性（「穩健性」），適應性衝突也很小（下文的穩健性和可塑性）。

穩健性（robustness）和可塑性（plasticity）

對4種不同水解酶（人血清對氧磷酶（PON1）、假單胞菌磷酸三酯酶（PTE）、蛋白酪氨酸磷酸酶（PTP）和人碳酸酐酶II（CAII））的研究表明，其主要活性對變化具有「穩健性」，而混雜活性較弱，更具「可塑性」。具體來說，選擇一個非主要反應（通過定向進化），最初不會減弱主要反應（因此它具有穩健性），但會極大地影響未選擇的反應（因此它們具有可塑性）。

來自微小假單胞菌（Pseudomonas diminuta）的磷酸三酯酶（PTE）在十八輪中進化為芳基酯酶（P-O到 C-O水解酶），特異性（K_M比值）發生了10⁹次變化，但大部分變化發生在初始階段，保留了未經選擇的殘留PTE活性，進化出的芳基酯酶活性增加，對於殘留PTE活性的喪失與芳基酯酶活性的提高有一點權衡。

這意味着根據IAD模型，進化時，專門的酶（功能單一）經歷了通用（功能多樣）的階段，然後又可能在基因重複後再次成為專門的酶，而混雜活動比主要活動更具可塑性。

重構酶（reconstructed enzymes）

酶進化的最新、最清晰易懂的例子是過去60年中生物修復酶技術的興起。其中氨基酸變化的數量非常少，為研究自然界中的酶進化提供了一個極好的模型。但通過使用現存的酶來確定酶家族是如何進化擁有缺點，就是將新進化的酶和同源酶相比較時不知道兩個基因分化之前祖先的真實身份。由於祖先重構，這個問題可以得到解決。祖先重建是由萊納斯·鮑林（Linus Pauling）和埃米爾·扎克坎德爾（Emile Zuckerkandl）於1963年首次提出的，是從一組基因的祖先形式推斷和合成一個基因The most recent and most clear cut example of enzyme evolution is the rise of bioremediating enzymes in the past 60 years. Due to the very low number of amino acid changes, these provide an excellent model to investigate enzyme evolution in nature. However, using extant enzymes to determine how the family of enzymes evolved has the drawback that the newly evolved enzyme is compared to paralogues without knowing the true identity of the ancestor before the two genes diverged. This issue can be resolved thanks to ancestral reconstruction. First proposed in 1963 by Linus Pauling and Emile Zuckerkandl, ancestral reconstruction is the inference and synthesis of a gene from the ancestral form of a group of genes, which has had a recent revival thanks to improved inference techniques and low-cost artificial gene synthesis, resulting in several ancestral enzymes—dubbed "stemzymes" by some—to be studied.

Evidence gained from reconstructed enzyme suggests that the order of the events where the novel activity is improved and the gene is duplication is not clear cut, unlike what the theoretical models of gene evolution suggest.

One study showed that the ancestral gene of the immune defence protease family in mammals had a broader specificity and a higher catalytic efficiency than the contemporary family of paralogues, whereas another study showed that the ancestral steroid receptor of vertebrates was an oestrogen receptor with slight substrate ambiguity for other hormones—indicating that these probably were not synthesised at the time.

This variability in ancestral specificity has not only been observed between different genes, but also within the same gene family. In light of the large number of paralogous fungal α-glucosidase genes with a number of specific maltose-like (maltose, turanose, maltotriose, maltulose and sucrose) and isomaltose-like (isomaltose and palatinose) substrates, a study reconstructed all key ancestors and found that the last common ancestor of the paralogues was mainly active on maltose-like substrates with only trace activity for isomaltose-like sugars, despite leading to a lineage of iso-maltose glucosidases and a lineage that further split into maltose glucosidases and iso-maltose glucosidases. Antithetically, the ancestor before the latter split had a more pronounced isomaltose-like glucosidase activity.

原始代謝（Primordial metabolism）

Roy Jensen in 1976 theorised that primordial enzymes had to be highly promiscuous in order for metabolic networks to assemble in a patchwork fashion (hence its name, the patchwork model). This primordial catalytic versatility was later lost in favour of highly catalytic specialised orthologous enzymes. As a consequence, many central-metabolic enzymes have structural homologues that diverged before the last universal common ancestor.

Distribution

Promiscuity is however not only a primordial trait, in fact it is very widespread property in modern genomes. A series of experiments have been conducted to assess the distribution of promiscuous enzyme activities in E. coli. In E. coli 21 out of 104 single-gene knockouts tested (from the Keio collection) could be rescued by overexpressing a noncognate E. coli protein (using a pooled set of plasmids of the ASKA collection). The mechanisms by which the noncognate ORF could rescue the knockout can be grouped into eight categories: isozyme overexpression (homologues), substrate ambiguity, transport ambiguity (scavenging), catalytic promiscuity, metabolic flux maintenance (including overexpression of the large component of a synthase in the absence of the amine transferase subunit), pathway bypass, regulatory effects and unknown mechanisms. Similarly, overexpressing the ORF collection allowed E. coli to gain over an order of magnitude in resistance in 86 out 237 toxic environment.

同源

Homologues are sometimes known to display promiscuity towards each other's main reactions. This crosswise promiscuity has been most studied with members of the alkaline phosphatase superfamily, which catalyse hydrolytic reaction on the sulfate, phosphonate, monophosphate, diphosphate or triphosphate ester bond of several compounds. Despite the divergence the homologues have a varying degree of reciprocal promiscuity: the differences in promiscuity are due to mechanisms involved, particularly the intermediate required.

Degree of promiscuity

Enzymes are generally in a state that is not only a compromise between stability and catalytic efficiency, but also for specificity and evolvability, the latter two dictating whether an enzyme is a generalist (highly evolvable due to large promiscuity, but low main activity) or a specialist (high main activity, poorly evolvable due to low promiscuity). Examples of these are enzymes for primary and secondary metabolism in plants (§ Plant secondary metabolism below). Other factors can come into play, for example the glycerophosphodiesterase (gpdQ) from Enterobacter aerogenes shows different values for its promiscuous activities depending on the two metal ions it binds, which is dictated by ion availability. In some cases promiscuity can be increased by relaxing the specificity of the active site by enlarging it with a single mutation as was the case of a D297G mutant of the E. coli L-Ala-D/L-Glu epimerase (ycjG) and E323G mutant of a pseudomonad muconate lactonizing enzyme II, allowing them to promiscuously catalyse the activity of O-succinylbenzoate synthase (menC). Conversely, promiscuity can be decreased as was the case of γ-humulene synthase (a sesquiterpene synthase) from Abies grandis that is known to produce 52 different sesquiterpenes from farnesyl diphosphate upon several mutations.

Studies on enzymes with broad-specificity—not promiscuous, but conceptually close—such as mammalian trypsin and chymotrypsin, and the bifunctional isopropylmalate isomerase/homoaconitase from Pyrococcus horikoshii have revealed that active site loop mobility contributes substantially to the catalytic elasticity of the enzyme.

Toxicity

A promiscuous activity is a non-native activity the enzyme did not evolve to do, but arises due to an accommodating conformation of the active site. However, the main activity of the enzyme is a result not only of selection towards a high catalytic rate towards a particular substrate to produce a particular product, but also to avoid the production of toxic or unnecessary products. For example, if a tRNA syntheses loaded an incorrect amino acid onto a tRNA, the resulting peptide would have unexpectedly altered properties, consequently to enhance fidelity several additional domains are present. Similar in reaction to tRNA syntheses, the first subunit of tyrocidine synthetase (tyrA) from Bacillus brevis adenylates a molecule of phenylalanine in order to use the adenyl moiety as a handle to produce tyrocidine, a cyclic non-ribosomal peptide. When the specificity of enzyme was probed, it was found that it was highly selective against natural amino acids that were not phenylalanine, but was much more tolerant towards unnatural amino acids. Specifically, most amino acids were not catalysed, whereas the next most catalysed native amino acid was the structurally similar tyrosine, but at a thousandth as much as phenylalanine, whereas several unnatural amino acids where catalysed better than tyrosine, namely D-phenylalanine, β-cyclohexyl-L-alanine, 4-amino-L-phenylalanine and L-norleucine.

One peculiar case of selected secondary activity are polymerases and restriction endonucleases, where incorrect activity is actually a result of a compromise between fidelity and evolvability. For example, for restriction endonucleases incorrect activity (star activity) is often lethal for the organism, but a small amount allows new functions to evolve against new pathogens.

Plant secondary metabolism

Anthocyanins (delphinidin pictured) confer plants, particularly their flowers, with a variety of colours to attract pollinators and a typical example of plant secondary metabolite.

Plants produce a large number of secondary metabolites thanks to enzymes that, unlike those involved in primary metabolism, are less catalytically efficient but have a larger mechanistic elasticity (reaction types) and broader specificities. The liberal drift threshold (caused by the low selective pressure due the small population size) allows the fitness gain endowed by one of the products to maintain the other activities even though they may be physiologically useless.

Biocatalysis

主條目：biocatalysis

In biocatalysis, many reactions are sought that are absent in nature. To do this, enzymes with a small promiscuous activity towards the required reaction are identified and evolved via directed evolution or rational design.

An example of a commonly evolved enzyme is ω-transaminase which can replace a ketone with a chiral amine and consequently libraries of different homologues are commercially available for rapid biomining (eg. Codexis).

Another example is the possibility of using the promiscuous activities of cysteine synthase (cysM) towards nucleophiles to produce non-proteinogenic amino acids.

Reaction similarity

Similarity between enzymatic reactions (EC) can be calculated by using bond changes, reaction centres or substructure metrics (EC-BLAST).

Drugs and promiscuity

Whereas promiscuity is mainly studied in terms of standard enzyme kinetics, drug binding and subsequent reaction is a promiscuous activity as the enzyme catalyses an inactivating reaction towards a novel substrate it did not evolve to catalyse. This could be because of the demonstration that there are only a small number of distinct ligand binding pockets in proteins.

Mammalian xenobiotic metabolism, on the other hand, was evolved to have a broad specificity to oxidise, bind and eliminate foreign lipophilic compounds which may be toxic, such as plant alkaloids, so their ability to detoxify anthropogenic xenobiotics is an extension of this.

參見

• Evolution by gene duplication
• Susumu Ohno
• Michaelis–Menten kinetics
• Protein moonlighting