研究酶的科研汪们都知道,无论是表达一种新酶,还是工程一个现有的酶,都需要对酶的一系列特性进行表征。小编上期已经介绍了酶的一些性质,点这里。后面小编会带大家逐渐了解如何测定酶的各种性质,不过小编毕竟水平有限,如有不当之处还请大家留言指正。这次,我们主要说说Km和Kcat。
Km、 Kcat、Vmax之间到底啥关系?我们先看上图。上图的横坐标是底物浓度,纵坐标是酶的反应速率。这个就是酶促反应动力学曲线了,可以用米氏方程来表征:
V = Vmax[S] / ( KM + [S]), 也可以写成V = Kcat[E]t[S] / ( KM + [S]),
公式中的V指反应速率,[S]是底物浓度,[E]t是酶的浓度,Km是一个常数,Kcat也是一个常数。这两个常数的意义我们后面再说。图中还有一个Vmax,这是最大反应速率,Vmax=Kcat[E]t.
从这个曲线我们可以知道,在底物浓度很低时,酶的反应速率会随着底物浓度的增加迅速增加。此时酶的活性位点的是空的,等着底物来结合,所以产物形成的速度由可利用的底物浓度决定。当底物的浓度增加时,酶的活性位点渐渐被底物饱和,此时底物形成的速率由酶的浓度决定,当活性位点被底物饱和以后,添加再多的底物都不会对酶促反应速率产生影响,此时达到最大酶促反应速率Vmax。
下面我们说Km和Kcat这两个常数:
Km(Michaelis constant,米氏常数)
Km是米氏常数(Michaelis constant),其实全称是米歇利斯常数,可能米歇利斯太难记了吧,所以大家都简称为米氏常数(为啥你们可以记住阿基米德原理,拉格朗日方程?小米同学应该会有点桑心)。米氏常数衡量的是酶与底物之间亲和力(affinity)的大小(这里指遵循米氏方程的酶)。Km等于酶促反应为最大反应速率一半时的底物浓度,其单位是mol/L,因此Km越小,表明酶进行反应所需的底物浓度越低,也可以说酶与底物之间亲和力越大。Km是一个常数,当pH,温度,离子强度不变时,Km是恒定的。
Km的重要性
Km是非常重要的参数,在酶的应用过程中,底物浓度选择常常需要参考酶Km。
1. 测定酶的活性
为了测定一个酶的活性,底物浓度应该怎么选择呢?我们通常是利用产物生成速率来衡量酶活性,在温度,pH等条件不变的情况下,产物的生成速率受到酶的活性,底物浓度的影响。我们为了测定酶的活性,自然应该使酶的活性本身作为唯一的变量,这就意味着底物的浓度必须非常高,使酶的活性位点饱和,确保酶催反应速率达到最大值Vmax。在实践中,底物的浓度常需要高出Km值的10-20倍。
2. 测定样品中底物的浓度
我们有时候会利用酶来测定其底物的浓度,比如利用荧光素酶测定ATP的浓度从而来测定细菌的数量(每个细菌内ATP数量是固定的,ATP越多说明细菌越多)。此时,我们需要保证底物的浓度是唯一的限制因素,底物的浓度必须要低于Km值,这样产物的生成速率会随着底物浓度的增加迅速提高,这就提高了检测底物的灵敏度。
Kcat
又叫转化数(turnover number),利用Vmax除以酶浓度进行计算。所以可以知道,Kcat衡量的是在最有优条件下酶催化生成底物的速率。为什么说是最优条件呢?因为此时反应速率最大(Vmax),酶的活性位点已经被底物完全饱和。Kcat是一个常数,其单位是1/s,也可以将Kcat理解成单个酶分子在一秒内转化底物的数量,或者单个酶分子转换一个底物分子所需的时间,因此Kcat也叫做转化数。
Kcat/Km
两个常数的比,当然也是一个常数。Kcat与Km的比是衡量一个酶催化效率的最重要参数。Kcat越大,Km越小,二者的比值越大。Kcat越大说明酶转化底物的速率越快,Km越小说明酶与底物之间亲和力越大。当酶有多个可利用的底物时,其对不同底物的催化效率可能差别很大。Kcat/Km就可以用来确定酶的最适底物,同一个酶对不同底物的Kcat/Km最大可相差100万倍。当利用蛋白质工程对酶进行改造时,不同突变型的Kcat/Km也是需要要测定的参数,用来表征酶催化效率的变化情况。
呀!不知不觉又写了这么多。Km,Kcat这么重要,应该怎么测量呢?等待小编的下篇文章吧。
バイアグラジェネリック йЂљиІ© – г‚·г‚ўгѓЄг‚№ гЃ®иіје…Ґ г‚їгѓЂгѓ©гѓ•г‚Јгѓ« жµ·е¤–йЂљиІ©
buy clindamycin generic – purchase indocin generic buy indomethacin generic
cost clavulanate – augmentin 1000mg drug cheap levoxyl
order augmentin online cheap – augmentin 625mg tablet buy synthroid for sale
order betamethasone 20gm cream – how to get monobenzone without a prescription monobenzone order
deltasone 40mg without prescription – omnacortil 20mg uk how to buy permethrin
where to buy accutane without a prescription – cheap avlosulfon deltasone 20mg oral
cyproheptadine generic – tizanidine drug purchase tizanidine
buy mobic pill – rizatriptan us toradol 10mg price
buy ozobax for sale – where can i buy ozobax buy piroxicam 20 mg without prescription
order diclofenac sale – purchase diclofenac online cheap cheap nimotop
buy pyridostigmine 60mg online cheap – mestinon generic buy generic azathioprine over the counter
buy generic rumalaya for sale – rumalaya generic amitriptyline over the counter
diclofenac 50mg tablet – aspirin canada aspirin 75 mg oral
mebeverine 135 mg brand – cilostazol canada buy pletal generic
buy calcort for sale – cheap alphagan buy brimonidine online
buy lactulose bottles – buy cheap generic lactulose order betahistine 16 mg
order cyclosporine sale – how to buy colchicine cost gloperba
order oxcarbazepine pills – buy pirfenidone medication levothroid cost
buy terazosin online cheap – flomax 0.4mg ca purchase dapoxetine
speman medication – purchase himplasia sale buy cheap generic fincar
finax tablets – buy finax medication buy generic uroxatral 10mg
noroxin drug – confido pill cheap confido sale
buy generic lasuna for sale – himcolin cost order himcolin pills
order generic atorlip – order bystolic 5mg online order bystolic online cheap
purchase gasex pills – buy gasex without a prescription purchase diabecon generic
order tenormin 50mg pills – order tenormin 50mg pill buy coreg no prescription
verapamil for sale online – diovan 160mg without prescription buy tenoretic sale
buy rogaine sale – generic dutas order propecia without prescription
purchase durex gel for sale – purchase durex gel for sale order zovirax
buy ondansetron sale – order detrol 1mg sale order requip 1mg generic
ascorbic acid order online – buy generic compro for sale compro pills
v1ezvx2mqggn13
http://www.nytonguesthouse.co.uk
spironolactone 25mg drug – naltrexone oral naltrexone 50 mg pills
buy disopyramide phosphate pill – buy disopyramide phosphate tablets order chlorpromazine 100mg
divalproex 250mg uk – oral amiodarone 200mg order topamax 100mg sale
Great read! The clarity and depth of your explanation are commendable. For further reading, here’s a useful resource: EXPLORE FURTHER. Let’s discuss!
nootropil without prescription – buy generic sustiva over the counter sinemet drug
buy feldene for sale – order rivastigmine 3mg exelon 3mg generic
brand monograph 600mg – buy cilostazol generic cheap pletal 100 mg
buy dramamine 50 mg pills – risedronate 35 mg pills actonel 35mg sale
buy forxiga 10 mg without prescription – doxepin online order order precose without prescription
order generic griseofulvin 250 mg – lopid online order purchase gemfibrozil pills
purchase cotrimoxazole for sale – bactrim sale how to buy tobrex
zovirax brand – zovirax uk duphaston 10mg cost
cost bisacodyl 5mg – liv52 10mg usa order liv52 20mg pill
buy aciphex 20mg online – aciphex drug domperidone for sale
fludrocortisone pills watch – prilosec pills well lansoprazole quiver
promethazine just – promethazine awkward promethazine issue
ascorbic acid cheek – ascorbic acid bob ascorbic acid peak
priligy able – priligy angle dapoxetine milk
valtrex pills jail – valtrex online depend valacyclovir online most
loratadine medication open – loratadine medication strain claritin grand
prostatitis pills third – prostatitis pills decent prostatitis medications defend
acne medication dart – acne treatment source acne medication expose
inhalers for asthma channel – inhalers for asthma joke asthma treatment fault
cenforce online carve – tadacip pills creak brand viagra dublin
priligy drift – udenafil newspaper cialis with dapoxetine rush
cialis soft tabs online flood – cialis super active online nothing1 viagra oral jelly aspect
brand cialis fellow – alprostadil defeat penisole snow
cenforce online lay – cialis for men brand viagra online cautious
brand cialis rider – tadora urge penisole lion
buy viagra professional myrtle – eriacta decision levitra oral jelly online ponder
dapoxetine fact – suhagra deceive cialis with dapoxetine fortune
rosuvastatin task – rosuvastatin online western caduet unit
zocor spy – zocor scar atorvastatin roger
nitroglycerin online – indapamide pills cheap diovan 160mg
lanoxin 250 mg over the counter – lanoxin tablet buy furosemide 40mg generic
buy nizoral tablets – buy itraconazole for sale order sporanox 100 mg pill
where can i buy famciclovir – valaciclovir sale valaciclovir generic
buy terbinafine for sale – buy grifulvin v generic purchase grifulvin v generic
buy rybelsus 14mg pill – glucovance ca buy DDAVP sale
order glyburide 5mg pills – glyburide 5mg usa buy dapagliflozin online cheap
cost of medrol – buy azelastine generic astelin 10ml sale
order albuterol 4mg without prescription – fexofenadine canada theophylline 400 mg usa
ivermectin 12mg online – ivermectin 6 mg without a doctor prescription buy cefaclor no prescription
cleocin 150mg uk – cleocin online buy chloromycetin for sale online
azithromycin price – tinidazole 500mg price order ciplox 500mg online cheap
buy amoxicillin generic – trimox 250mg tablet cipro 1000mg cheap
order augmentin online cheap – bactrim over the counter cipro 500mg us
oral hydroxyzine 10mg – order atarax 10mg generic purchase endep pill
buy quetiapine sale – brand venlafaxine 75mg cheap eskalith generic
clomipramine over the counter – purchase paroxetine for sale cost sinequan
buy clozapine 100mg generic – order glimepiride pills order pepcid online
metformin 500mg without prescription – generic glucophage purchase lincocin pill
furosemide medication – purchase capoten for sale generic capoten 25 mg
buy flagyl without prescription – order terramycin 250mg without prescription purchase azithromycin without prescription
purchase ampicillin generic buy amoxil generic cheap amoxil pills
purchase valacyclovir generic – how to buy nemasole zovirax pills
ivermectin 6 mg online – suprax medication tetracycline 250mg for sale
how to get metronidazole without a prescription – order cefaclor 250mg without prescription zithromax 500mg generic
buy generic ciprofloxacin online – buy erythromycin cheap erythromycin uk
ciprofloxacin uk – purchase cephalexin for sale order augmentin 625mg generic
order ciprofloxacin – buy keflex online cheap amoxiclav
order atorvastatin 40mg online buy lipitor tablets atorvastatin 80mg tablet