ISOENZYMETri Rini Nuringtyas

Within a single species exist several different forms of enzyme catalyzing the same reaction

Enzim-enzim tersebut berbeda satu dengan yang lain berdasarkan : Urutan asam amino Modifikasi kovalen pada beberapa asam amino

penyusunnya (phosphorilation of serine hydroxyl group)

Struktur 3-D (conformational change)

Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but

catalyze the same chemical reaction

Term isoenzyme terbatas pada enzyme yang secara genetis telah berbeda pada urutan asam aminonya.

Sehingga tidak berlaku untuk enzim yang berbeda karena mengalami modifikasi dan mempunyai urutan asam amino yang sama.

Mengapa kita perlu isoenzyme?

Untuk pengaturan metabolisme Untuk memenuhi kebutuhan suatu jaringan Untuk memenuhi kebutuhan pada fase

perkembangan tertentu sel.

Beberapa enzim dikenal mempunyai isoensim. Antara isoensim satu dengan yang lain mungkin berbeda pada: Kecepatan turn-over nya Kemampuan untuk diatur Keberadaan pada organ yang berbeda atau organel yg

berbeda Fase perkembangan yang berbeda

Keberadaan suatu isoenzim tertentu, tidak menutup kemungkinan isoenzim lainnya tetap ada. Hanya berbeda pada jumlah / kuantitasnya

Isoenzim vs alloenzimThe gene products (enzymes) of the different gene

loci (pseudoalleles) are called isoenzymes.

isozymes (or isoenzymes) are isoforms (closely related variants) of enzymes.

Suatu organisme yang diploid, maka akan terdapat dua alel untuk setiap gene nya.

A dan a Suatu gene menentukan formasi suatu rantai

polipeptida

Apabila enzim terdapat dlm btk heterozigot : Aa , maka dua rantai polipeptida akan terbentuk.

Dan kedua rantai polipeptida tersebut mungkin berbeda dlm turnover rate maupun pd saat dipisahkan dg elektroforesis

A mungkin menghasilkan btk aktif,

a mungkin menghasilkan btk tidak aktif

Polypeptides (enzymes) yang dikode oleh allel yang berbeda alloenzymes.

allozymes represent enzymes from different alleles of the same gene,

isozymes represent enzymes from different genes whose products catalyse the same reaction,

Bagaimana cara membedakan alloenzyme dan isoenzyme?

Gel electrophoresis saja tidak dapat membedakanallo- and isoenzymes.

Studi mengenai alloenzyme memerlukan individu yg berbeda pada species yg sama. Sel yang sama harus dibandingkan dan harus mengikuti segregasi Mendel.

Untuk studi isoenzyme pada dasarnya satu individu cukup krn setiap individu mempunya pattern distribusi yg sama

Alloenzymes adalah marker yg sangat bagus untuk kajian genetika populasi.

Isoenzymes penting untuk belajar fisiologi perkembangan suatu spesies

Contoh isoenzyme : glukokinase. Suatu variant / turunan dari heksokinase. Ensim ini berbeda dgn heksokinase karena tidak dihambat oleh glucose 6-phosphate. Jadi mempunyai pola pengaturan dan affinitas yg lebih rendah dengan glukosa (Km ?).

allows it to serve different functions in cells of specific organs, such as control of insulin release by the beta cells of the pancreas, or initiation of glycogen synthesis by liver cells. Both of these processes must only occur when glucose is abundant, or problems occur.

Isoenyzyme analysis can determine the species identity of your cell line and determine if your cell line has been contaminated by a cell line of a different species.

Plants contain multiple isoenzymes of GS, some located in the cytoplasm (=GS1) and another in plastids (= GS2).  All plants examined contain at least 3 GS1 isozymes, but only one nuclear encoded GS2 isoenzyme.  

The technique of allozyme electrophoresis is one of the most common approaches used to identify discrete stocks of fish. It is a powerful tool for identifying stocks of some species, and we use this approach extensively in our work with sockeye salmon, chinook salmon, pink salmon, and chum salmon. Each of the 10 lanes represents the enzymes products from the genes of a single fish. Samples of fish tissues must be frozen immediately upon collection to preserve the enzymes for analysis in the lab.

Picture to the right: Variations of the leucine-amino-peptidase (LAP) in snails. Each row corresponds to a probe of one animal each. The patterns are specific for every individual.

Nomenclature of isoenzyme

it is recommended based on : the extent of migration in an applied

electronic field using electrophoresis

not recommended to name isoenzyme based on : tissues distribution (example brain type or muscle type) since distribution can vary between different species

Isoenzyme are numbered : starting with the species having highest mobility towards anode

Lactate dehydrogenase

occurs in a variety of forms consider as isoenzymeis coded for by 2 genes - it has 2 isoenzymes.

One is the heart = H form main isoenzyme in the heart.The other is the muscle = M form main isoenzyme in muscle.

Picture to the left: Isoenzyme pattern of lactate dehydrogenase (LDH) in heart tissue of embryonic, iuvenile and adult mice. The six rows show the pattern -9, -5, -1 days before birth as well as +12 and +21 days after birth and in adult mice.

LDH-1 predominant in heart, erythrocyte, brain and kidney

LDH-3 predominant in leucocytes, adrenal

and thyroid

LDH-5 predominant in skeletal muscle and

liver

The subunits (H and M) very similar in size and shape but

differ in charge

The H isoenzyme has more aspartate while The M isoenzyme has more lysine

Character: differential heat stability : LDH-4 & LDH-5 show greater

heat stability

LDH-1 is more sensitive to inhibition by excess pyruvate than LDH-5

Pyruvate is converted to lactate for an immediate ATP reward.Get less ATP, but don’t need oxygen

LDH-Hlactate pyruvate LDH-M

Therefore, heart tissue has mostly H4 isoenzymes, muscle has mostly M4 isoenzymes.

each organ has its own unique pattern of the five LDH isoenzymes is used to diagnose internal injuries, because a

damaged organ releases LDH into the bloodstream.

Substrate concentration dependence LDH-5 is maximally active in the presence of 250 mM

lactate, LDH-1 isoenzyme for optimal lactate concentration 10

mM,

a 50% inhibition of LDH1 activity when it is assayed using 250 mM lactate as a substrate.

By measuring LDH activity using both 10 and 250 mM lactate concentrations, the calculated ratio of the two activities helps to judge whether the sample contains

LDH1 or LDH5 isoenzyme.